Sundae POSTERS 626-627.05

drop to a low value of approximately 10 events/msec during strongstimulation, which depletes the readily-releasable pool of vesicles.This rate is as expected for a pool recovery time constant of about300 msec, assuming that the steady state release representsvesicles, which are newly recruited to the pool and immediatelyreleased.

Exocytosis & Endocytosis

627.01-Pos Board # B483MODELING OF CALCIUM DEPENDENT PRESYNAPTICRELEASE SYSTEMS.Najl V. Valeyev; Kazan State University, Kremlevskaja - 18,Kazan, Tatarstan Republic 420008 Russian FederationCalcium signaling proteins display a large variability concerningtheir affinity, specificity, and kinetics. The question is how thenecessary selectivity of activation of diverse targets can beregulated by calcium concentration. We have developed themathematical model of nonequilibrium Ca2+ distribution in thepresynaptic terminal. The model has been created in analogy withmodel previously developed by Markram H et. al. 1998 fordendritic like compartment. To explore the possible implications ofnonequilibrium calcium dynamics in presynaptic terminal, wesimulated calcium influx, buffering and binding by proteins crucialfor neurotransmitter release. The results suggest that thepresynaptic calcium signaling system may activate differentrelease systems selectively by different calcium accumulating inbuffers and binding by proteins far from equilibrium.

627.02-Pos Board # B484SECRETION CAN BE EVOKED BY ACTIVATION OFEXOGENOUS CALCIUM CHANNELS IN A MOUSEPHEOCHROMOCYTOMA CELL LINE.Amy B. Harkins', Anne L. Cahill', Arthur S. Tischler2, Aaron P.Fox'; 'University of Chicago, 947 E. 58th Street, Chicago, IL60637, 2Tufts University School of Medicine, Boston, MA 02111We have characterized a recently-developed immortalized mousepheochromocytoma (MPC) cell line to determine whether thesecells can be used as a model cell for secretion. MPC cells containsmall synaptic-like vesicles as determined by electron microscopy.Additionally, MPC cells contain both syntaxin I andsynaptotagmin I as determined by immunocytochemistry.However, when single, untreated MPC cells were patch-clamped inthe whole-cell recording configuration, the cells expressed a Na+current (n=88) and little or no endogenous Ca2+ current. Of the 88cells, only 19 had a small endogenous Ca2' current which averaged78 ± 8 pA (mean ± SEM). No measurable secretion was observedin MPC cells (n=27). Cells treated with either NGF (n=6) or EGF(n=8) did not exhibit an increase in either Ca2+ current amplitudeor secretion. MPC cells were transiently transfected with Ca2+channel subunits (a,o, 2, and a28). Stimulation with a train of 5depolarizations from a holding potential of -80 mV to +20 mV(200 ms test pulse, 50 ms interpulse) produced a Ca2+-influx of 880x 106 ions (+ 96 x 106 ions, n=15), and peak secretion of 376 fF (±75 fF, n=15). o-Cgtx GVIA (1 pM) completely blocked thetransfected a,B Ca2+ current (n=5), as well as secretion (n=3).Thus, previously unavailable MPC cell lines can serve as surrogatemodels of secretion, complementing existing cell lines such asPC12 cells. Furthermore, the MPC cells are readily transfectedand may be particularly useful for studies in which a mouse cellline would be advantageous. Supported by grants to APF(NS33826-04) and AST (NS37685).

627.03-Pos Board # B485A CALCIUM CLAMP FOR STUDYING EXOCYTOSIS ANDENDOCYTOSISJames Dunning, Kevin D Gillis; University of Missouri -Columbia, Research Park, Columbia, MO 65211Photorelease of Ca from caged compounds has been widely used tostudy Ca-activated cellular processes such as exocytosis andendocytosis. However, photolysis of caged compounds is usuallydone in an "open loop" configuration without tight control of theamplitude and time course of the resulting free Ca concentration([Ca]). We have implemented a feedback control system using amonochromator both to photolyze the cage and excite a fluorescentdye to report [Ca]. We have been able to elevate [Ca] from -150nM to 1 jM in about 200 ms and maintain [Ca] within -+1-5% ofthe desired level indefinitely. Complex [Ca] stimulus patterns arepossible. In another configuration, [Ca] can be elevated within - 1ms with a flash lamp and then maintained at a constant level usingthe monochromator. In our system, the Ca cage NPEGTA ismaximally photolyzed at a wavelength of -360 nm. In order tominimize photorelease while measuring [Ca], we excite the Caindicator dye bisfura2 at 330 and 410 nr. The system could befurther improved if a high-affinity, ratiometric Ca indicator excitedat long wavelengths could be found. We believe that the Ca clampwill be particularly useful for studying both the priming andtriggering of exocytosis at [Ca] levels of - 1 jiM where the rate ofexocytosis is slower than the response time of the Ca clamp.

627.04-Pos Board # B486CALCIUM ELEVATION IS REQUIRED FOR VESICLEPOOL MOBILIZATION AT HIPPOCAMPAL SYNAPSES.Timothy Aidan Ryan, Thomas Fernandez-Alfonso; WeillMedical College Cornell, 1300 York Ave, New York, NY 10021Using multiple optical tracing approaches of synaptic vesicletraffic (FM 1-43 and synaptopHluorins) we show that prolongedand repeated action potential stimulation in minimal externalcalcium ([Ca2k],,) leads to turnover of only - 10-15 % of the totalrecycling pool. Stimulation in intermediate levels of [Ca2i]. leadsto larger but stil incomplete pool turnover compared to that whichoccurs in elevated [Ca2'].3. Our data indicate that in addition to thecalcium sensor for exocytosis, a calcium-sensitive switch controlswhether vesicles in the reserve pool make transit to the readily-releasable pool to participate in vesicle recycling. Finally our dataindicate that vesicles that undergo exocytosis during minimalcalcium elevation become functionally mixed within the reservepool upon recycling.

627.05-Pos Board # B487A TECHNIQUE FOR MEASURING MEMBRANECAPACITANCE DURING A DEPOLARIZING STIMULUSPeng Chen, Kevin D Gillis; University of Missouri - Columbia,Research Park, Columbia, MO 65211Depolarization-evoked exocytosis is often inferred from thechange in membrane capacitance (Cm) measured at hyperpolarizedintervals before and after a depolarizing pulse. Cm measurementsare usually not attempted in excitable cells during the depolarizedpulse because the activation of nonlinear time-dependentconductances invalidates the simple 3 component equivalentcircuit of the cell. This necessitates complex pulse protocols toindirectly infer the time course of exocytosis during adepolarization-evoked influx of Ca. We have found that theaddition of one additional time constant (2 parameters) to theequivalent circuit is able to model both the nonlinear, time-dependent Ca conductance and the current due to the motion of"gating" charges of voltage-dependent ion channels. We haveused a broadband "pseudo-random binary sequence" voltagestimulus and a nonlinear least-squares algorithm to estimate the 5model parameters during membrane depolarization and find that

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the Cm estimate is only slightly biased by a depolarization-activated Ca current several hundred pA in amplitude. The noiseof Cm measurements during the depolarization is near theoreticalestimates and may allow the measurement of the size of the"immediately releasable pool" of vesicles during a singlemembrane depolarization.

627.06-Pos Board # B488QUANTITATIVE IMAGING OF NAD(P)H IN GLUCOSE-STIMULATED B-CELLS.Jonathan V. Rocheleau, David W. Piston; Vanderbilt University,702 Light Hall, Nashville, TN 37232-06153-cells secrete insulin in response to glucose by an increase in

metabolic flux, which yields an associated increase in reducednicotinamide dinucleotide (phosphate) [NAD(P)H]. The relativerole of glycolytic and tricarboxylic acid (TCA) cycle metabolismin driving secretion remains unclear. Using 710 nm laser light,two-photon excitation microscopy can be used to assayquantitatively NAD(P)H levels. The NAD(P)H fluorescenceshows a dark nucleus (low [NAD(P)H]), moderately brightcytoplasm (medium [NAD(P)H]), and bright punctatemitochondria (high [NAD(P)H]). It is thus possible to separate thecytoplasm and mitochondria signal. Glucose-stimulation causes a>1.5 fold increase in both cytoplasmic and mitochondrial intensitywithin 20-40s. Combining the spatial resolution of the subcellularcompartments with specific inhibitors of the malate-aspartateshuttle or the mitochondrial-pyruvate transporter, the NAD(P)Hlevels observed in mitochondria can be associated with glycolyticproducts or direct activation of TCA metabolism. Both inhibitorslower the mitochondrial-NAD(P)H response without changing thetime to reach a plateau. These results suggest that the TCA cycleis active very early in the glucose response, and indicate that theTCA cycle may be necessary for insulin secretion.

627.07-Pos Board # B489DYNAMICS OF SECRETORY GRANULES IN LIVINGNEUROENDOCRINE CELLS VISUALIZED BY TOTALINTERNAL REFLECTION FLUORESCENCEMICROSCOPYJean-Baptiste Manneville, Lindsay McGuinness, Abdul K Sesay,Paul Skehel, lain C. A. F. Robinson, Michael A Ferenczi; NationalInstitute for Medical Research, The Ridgeway, Mill Hill, London,NW7 1AA United KingdomWe used total intermal reflection fluorescence microscopy(TIRFM) to study the dynamics of secretory granules in livingneuroendocrine cells derived from rat pituitary tumors (growthhormone producing cells, GC cells). Secretory granules werefluorescently labelled using a GFP construct of the v-SNAREprotein synaptobrevin (VAMP-GFP). Real-time tracking ofindividual granules in unstimulated cells enables us to distinguishbetween two types of dynamic behaviours: slow diffusive-likemotions with diffusion coefficients of the order of 104-410i Jm2/sand long-range directed motions with velocities of the order of 0.2-2 tm/s. We also used a GFP construct of human growth honnone(hGH-GFP) to study the role of a dominant negative mutation ofhGH, known to inhibit the production of GH in patients sufferingfrom severe isolated GH deficiency. Our results show a disruptionof the packaging of GH into granules in cells expressing themutant GH compared to wild-type. TIRFM imaging allows us tocompare quantitatively the dynamics of hGH-GFP containinggranules in mutant or wild-type GH expressing cells followingstimulation of release.

627.08-Pos Board # B490APPLYING OPTICAL TWEEZERS TO STUDY POST-FUSION MECHANISMS OF EXOCYTOSIS.Wolfgang Singer, Stefan Bermet, Monika Ritsch-Marte, ManfredFrick, Thomas Haller, Paul Dietl; University of Innsbruck,Mullerstr. 44, Innsbruck, Tirol A-6020Exocytosis of lamellar bodies, the storage form of pulmonarysurfactant, by alveolar type II cells is a prerequisite to lowersurface tension at the air-liquid-interface in the lung. The post-fusion mechanisms of secretion and extracellular transformation ofreleased surfactant are poorly understood issues. However, severalindependent lines of observation indicate that in type II cells therelease of the vesicle contents is controlled by the size of the fusionpore. We have used a self-designed dual-beam optical tweezerssetup with pico-Newton force calibration to investigate themechanical properties of fused lamellar bodies by applyingcontrolled pulling forces. In particular, we have been able to pulllamellar bodies after fusion with the plasma membrane, movingsurfactant through fusion pores into the extracellular space. It waspossible to expand "threads" of surfactant to the considerablelength of several cell diameters without detaching them from thecells. Our observations support the notion that the fusion pore inthe type II cell is a narrow site by which the rate of vesicle releaseis limited. Studying the visko-elastic properties of surfactant usinglaser tweezers allows to gain insight into the post-secretoryextracellular organization of surfactant and into the biophysicalforces involved in the process of release.

This work was supported by the Austrian Science Fund (FWF-Projects P14263-MED and P12974-MED)

627.09-Pos Board # B491CONTROL OF SPERM EXOCYTOSIS BY STEROLS.Nicholas L. Cross; Oklahoma State University, 264 McElroy Hall,Stillwater, OK 74078Ejaculated mammalian sperm must complete a final maturation,termed capacitation, before they can undergo exocytosis andfertilize eggs. An early, obligatory step in capacitation is loss ofthe sperm sterols, cholesterol and desmosterol, that inhibitexocytosis by unknown mechanisms. To provide information onhow these sterols work, we measured the inhibitory activities ofstructural analogs. Human sperm were incubated 24 hr in mediawith sterol-containing phosphatidylcholine liposomes and thenexposed to progesterone, an inducer of acrosomal exocytosis. Inparallel samples, sperm lipids were extracted and sterols wereassayed by gas chromatography. We first assessed the relativeinhibitory activities of cholesterol and desmosterol; their ICsoswere not significantly different. The inhibitory activity of ergasta-5,7,9 (11),22-tetraen-31-ol was similar to cholesterol. Cholesterolethyl ether and coprostanol were less inhibitory than cholesterol,and 5a-androstane-30-ol was more inhibitory. These resultssuggest that the inhibitory activity may require a planar ringstructure and a 3-hydroxyl group, and that considerable variation inthe aliphatic tail and degree of unsaturation of the ring structure istolerated. Supported by NIH grant HD 30763.

627.1-Pos Board # B492FAST EXOCYTOSIS BY THE CHICK COCHLEAR HAIRCELLMaria Spassova, James C. Saunders, Thomas D. Parsons;University of Pennsylvania, 382 West Street Road, KennettSquare, PA 19348We have utilized changes in cell membrane capacitance (AC.,,) as aproxy for neurotransmitter release to study presynaptic cellular andmolecular specializations. We previously demonstrated at the haircell-afferent fiber synapse a large pool of releasable vesicles(-5000) that exhausted with a time constant of 600 ms. This large,slowly released pool is several times larger than the number of

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vesicles expected to be found in close proximity to theplasmalemma at hair cell release sites. Recent improvements inour recording techniques have allowed us to resolve another,smaller, faster kinetic component of exocytosis. Shortdepolarizing pulses ranging from 50 to 200 ms resulted incapacitance jumps whose amplitude saturated with a time constantof 50 ms at an amplitude of -35 fF (-1000 vesicles). Longerdepolarizing pulses activated exocytosis of the larger, slowlyreleased pool (500 ms, ACm=126 +/- 14fW). Although this largeincrease in ACm was completely blocked by 2.5 mM of the fastCa2+ chelator BAPTA (6 +/- 6fW), it was only partially blocked by2.5 mM of the slow Ca2chelator EGTA (37 +/- 7 fF). Theseexperiments suggest the presence of a small fast pool of vesiclesthat is resistant to buffering by slow Ca2+ chelators, and maycontribute to the ability of the hair cell-afferent fiber synapse tomeet the phasic requirements of audition.

627.11-Pos Board # B493NON-STATIONNARY FLUCTUATION ANALYSIS OFMEMBRANE CAPACITANCE YIELDS INFORMATIONON SECRETORY PROCESSAlexandre Bourque-Viens', Remy Sauv62, Marcel D. Payet';lUniversite de Sherbrooke, 3001, 12e avenue nord, Sherbrooke,Quebec JIH 5N4 Canada, 2GRTM, ddpartement de physiologie,Universite de MontrdalAn assessment of the process responsible for the modulation of theexcitation-secretion coupling is of interest for studyingphysiological mechanisms such as neurotransmission,neuromodulation, facilitation, LTD, LTP, etc. Previous works onsecretion using fluctuation analysis of membrane capacitance wereoriented on average vesicle size evaluation [T. Moser & E. Neher(1997), PNAS, 94:6735-6740.1 In this study, the fluctuationanalysis method was optimized to produce the average andvariance from an ensemble of a low number of repetitions (3 to10). Exploratory experiments conducted on neuro-glioma hybridNG 108-15 cells tend to support the validity of the approach.Relations between variance and mean of ensemble compatible witha multinomial distribution were observed. Preliminaryinterpretation points to the presence of a principal componentassociated with the fusion of vesicles and additional componentsthat could account for membrane recuperation. Data were fitted toa model of vesicle trafficking at the cell membrane, i.e. fusion andrecuperation. This transposition of fluctuation analysis to thetreatment of membrane capacitance records offers new tools tostudy the mechanisms that come into play in modulatingexcitation-secretion coupling.This work was supported by grants from CMRC (#MOP-37912).

627.12-Pos Board # B494QUANTITATIVE MOTION ANALYSIS OF INSULINGRANULES IN LIVING PANCREATIC CELLSJuris Galvanovslds, Erik Renstrom, Sebastian Barg, RositaIvarsson; Lund University, Institute of Physiology andPharmacology, Solvegatan 19, Lund, 223 62 SwedenWe used confocal laser scanning microscopy to tracksimultaneously multiple labelled insulin containing granules inINS-1 cells. It was found that these granules present variouspatterns of movement prior to be released into extra-cellular space.Obviously essential information about conditions and processes

inside a cell during insulin release is contained in these patterns,and it can be extracted by applying appropriate methods of dataanalysis. For example, the increase of surrounding temperatureenhanced both the intensity of Brownian-like movements and theprobability to observe saltatory jumps. At the same time thevelocities achieved by granules during saltatory movements were

not affected. In this investigation we apply statistical methods(based on the theory of Wiener processes) to characterize thesemovements under various conditions. The results show that

according to these methods granules can be divided into severalpools that differ by their statistical characteristics. The largest poolcontains granules that perform Brownian-like movements; severalsmaller pools consist of granules that are involved into asystematic drift of various degrees. We conclude that thesedifferences reflect the various stages of granule maturation and aswell as different protein interactions prior to granule exocytosis.

627.13-Pos Board # B495KINETICS OF HOMOTYPIC CORTICAL VESICLEFUSION.Jens R. Coorssen', Joshua Zimmerberg2, Paul S. BlankZ; 'NationalInstitutes of Health and University of Calgary, Calgary, AlbertaCanada, 2National Institutes of Health, Bethesda, MD 20892-1855Isolated cortical vesicles (CV), the fully primed and release-readysecretory organelles of sea urchin eggs, can be centrifuged intocontact and the response to increasing free calcium concentrationsassayed using established end-point fusion assays. The results ofsuch studies have indicated great similarity between calcium-triggered exocytosis in the isolated planar oortex (endogenouslydocked CV at the plasma membrane) and the homotypic fusion ofisolated CV (Coorssen, et al., 1998, J. Cell Biol. 143, 1845-1857).We have extended further the comparison of these two systems toinclude the kinetics of fusion. In response to elevated free calciumconcentrations, CV-CV fusion proceeds in a manner comparable tothe kinetics observed for exocytosis in the isolated planar cortex.The kinetics of fusion are described by our mathematical analysisthat relates an increase in the free calcium concentration withincreases in the average number of participating fusion complexes(Vogel, et al., 1996, J. Cell Biol. 134, 329-338). Protease andthiol-specific treatments of CV prior to contact and calciumtriggering alter the kinetics of fusion. Protease treatment canproduce a reduction in the rate of fusion in the absence of changesin the average number of fusion complexes. These results supportour previous hypothesis (Coorssen, et al., 1998) that bothmodulatory proteins and fusion proteins are required for efficientcalcium-triggered membrane fusion in vivo.

627.14-Pos Board # B496CAPACITATIVE CA2+ INFLUX REGULATES MEMBRANETRAFFICKING IN HUMAN T CELLS.Alla F. Fomina, Michael D. Cahalan; Dept. of Physiol. andBiophysics, UCI, Irvine, CA 92697.Although CRAC channels play a crucial role in early stages oflymphocyte activation, their role in later phases is largelyundefined. We have described upregulation of CRAC channelfunctional expression in activated human T cells (Fomina et al,2000. J.Cell Biol. 15:1435-1444). Concentrations of membranesignaling proteins at the site of contact with antigen have beenreported, although underlying mechanisms remain unknown. In

this study, we compared membrane turnover in resting and PHA-activated human T cells. Membrane trafficking was visualized byconfocal imaging of FM1-43 fluorescent dye. Bright spots ofFM1-43 fluorescence were observed on the periphery of activated,but not resting T cells. These "hotspots" colocalize with rafts ofmembrane protein identified by FITC-labeled cholera toxin. Therate of constitutive membrane internalization was substantiallyhigher in activated T cells. Capacitative Ca2e influx triggered bythapsigargin increased the rate of dye accumulation. In addition,formation of new "hotspots" of FM1-43 fluorescence was observedfollowing Ca2+ entry. The data suggest that constitutive membraneturnover is upregulated during T cell activation. Membraneturnover can be modulated by external stimuli which affect Ca2+homeostasis. Ca2' stimulated membane trafficking could producefast changes of membrane composition and insertion of membraneproteins. Supported by NIH grants NS-14609, GM-41514 andAHA grant 0030275N.

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Polymer Physical Chemistry

628-Pos Board # B497WATER-MEDIATED SITE-SPECIFIC CATION BINDINGTO DNA: MOLECULAR DYNAMICS CALCULATIONS.George R Pack, Gene Lamm, Eric C. Magnuson; Univ. ofLouisville, 2320 Brook St., Louisville, KY 40292Molecular dynamics calculations of mono- and divalentcounterions near canonical B and crystal models of[d(CGCGAA'ICGCG)J2 were performed to investigate sequencespecific cation-DNA binding. A single divalent ion andneutralizing monovalent ions with starting positions chosen fromhigh occupancy regions based on previous Monte Carlocalculations. Trajectories were analyzed on the basis of cationposition and motion, specific hydrogen bond patteras and duration.The divalent cation maintained its full octahedrally coordinatedhydration shell. Specific cation-water-DNA hydrogen bonds lastedfor about 300 ps with two or four bonds often presentsimultaneously. When significant migration of the cation occurred,the motion was usually that of "rolling"' along a groove as if thehydrated ion were handed off from one DNA proton donor oracceptor to the next. Only occasionally would the ion actuallybreak all hydrogen bonds, diffuse out into the DNA hydration shelland reenter the groove somewhere else. To investigate the extentof cation-i interactions between a cation and a nucleotide base, anoctahedrally hydrated magnesium ion and a C or G base wereextracted from the crystal structure. All hydrogen atoms were thenoptimized at the B3LYP/6-31G* level with single-point energyMP2 calculations performed with the 6-31 1++G**basis set.Interaction energies obtained were mainly electrostatic and lessthan that of a hydrogen bond, suggesting that, at least in this case,cation-i interactions are not determinants in cation-DNA bindingspecificity.

629-Pos Board # B498EXPONENTIALLY VARYING CONCENTRATION OFSOLUTES NEAR THE SURFACE OFHYDROXYPROPYLCELLULOSE.John K Chik, Don C. Rau, V. Adrian Parsegian; NICHD/NIH,Rockville, MD 20892-5626Small solutes and large molecule osmotic stress control theswelling of weakly ordered pellets of hydroxypropylcellulose(HPC), a neutral, water-soluble derivative of cellulose. Changes inthe x-ray Bragg peak position when KF, KCI, KBr, glycerol,methylglucoside or betaine are added to solutions of known PEGosmotic pressure, show osmotic behavior, attributed to their partialexclusion from HPC. This partial exclusion exponentially decaysas a function of separation between HPC surfaces withcharacteristic decay lengths of d,,w-34A, similar to the 4ydobserved for hydration forces. Exclusion also appears independentof bath concentration (<I.5M). Although the decay lengths weresimilar, the amplitudes of exclusion were strikingly different. ForKP, KCI, and KBr, the amplitudes of exclusion follow the anionordering of the Hofmeister series and is proportional to anionsolvation entropy. Ion interaction with HPC is mainly throughextended hydration shells and not strictly steric. Weak temperaturedependence is observed not only for amplitudes but also decaylengths of exclusion for the salts. However for glycerol,methylglucoside and betaine only the amplitudes of exclusionchanges noticeably with temperature.

630-Pos Board # B499THE EFFECTS OF SUBPHASE IONIC STRENGTH ANDELECTROSTATIC INTERACTIONS AT THE PROTEIN-PROTEIN CONTACT IN TWO-DIMENSIONALSTREPTAVIDIN CRYSTALSGrady R. Blacken', Thomas Z. Armell, Patrick S. Stayton2, ToddC. Edwards'; 'Whitman College, 345 Boyer Ave., Walla Walla,

WA 99362, 2University of Washington, Univ. of Washington Box357962, Seattle, WA 98195We examined the effect of subphase ionic strength andintermolecular interactions at the crystal contact of two-dimensional streptavidin crystals grown beneath lipid monolayers.Using Brewster angle microscopy, variations in crystalmorphology were quantified relative to crystals formed in a pH 7,50mM PBS 500mM NaCI buffer. Crystals grown in varying NaClconcentrations all exhibited the typical X morphology, howeverthose grown around 500mM NaCl had a greater aspect ratio thanthose grown around OmM NaCl. Interestingly, crystal formationwith 100-480mM NaCl buffer appears to have been inhibited. Tofiurther investigate the effect of intermolecular interactions oncrystal morphology, we engineered several streptavidin mutantsusing site-directed mutagenesis. To probe the role of arginine 103and glutamic acid 101 at the P1 contact, we created separatemutations (R103A, ElOlV) to characterize a potential salt bridgeat the contact. Additionally, we replaced alanine 35 with anegatively charged residue (A35E) to try to create a salt bridgeacross the contact with arginine 53. Mutants designed to alter thespace group of the crystals that formed under specific buffer pHconditions have been purified and are currently being crystallized.

631-Pos Board # BSOOFREE SOLUTION ELECTROPHORESIS OF LYSOZYMECHARGE MUTANTSJennifer A. Durante, Thomas M. Laue', Thomas P. Moody',Stuart A. Allison2; 'University of New Hampshire, 46 CollegeRoad, Durham, NH 03824, 2Georgia State University, Atlanta,Georgia 30303Accurate values of the molecular charge on a macroion in solutionare hard to obtain. This is because in the process of measurementby electrophoresis, coupling between the flows of macroion andsolvent ions give rise to additional frictional forces beyondhydrodynamic ones. As a means of assessing these effects,membrane confined electrophoresis (MCE) experiments with T4charge mutants are being conducted. The charge changes are aresult of substitution of certain arginine and lysine residues withglutamic acid thus lowering the formal charge at neutral pH from+9 to +1 while maintaining the same hydrodynamic frictionalcoefficient. The molecular charge necessary in boundary element(BE) modeling to account for experimental mobilities is comparedto predicted formal charges of the mutants.

632-Pos Board # B501OSMOTIC PROPERTIES OF DNA: CRITICALEVALUATION OF COUNTERION CONDENSATIONTHEORYPer Lyngs Hansen, Rudi Podgornik, and V. Adrian Parsegian;LPSB/NICHD, NIH, Building 12A, Room 2041, Bethesda, MD20892-5626The osmotic coefficient of dilute B-DNA solutions in near-distilled water and in dilute salt solutions can deviate by 100 %from predictions based on the line-charge 'counterioncondensation' theory of Oosawa and Manning. We criticallyevaluate 'condensation' theory by contrasting its predictions withthose of the salt-free Lifson-Katchalsky cell model with Donnan-type corrections for salt effects. Remarkably, the cell modelpredictions capture quite well the osmotic properties of B-DNAover the entire DNA concentration range from 10mM to 1 M, bothunder 'zero' salt conditions and in 2 and 10 mM NaCl. By contrast,over no concentration range does Oosawa-Manning theorydescribe these properties well. Oosawa-Manning theory fails, notonly because it ignores some nonlinear electrostatic effects, butalso because as a line-charge theory, it is only valid in theinfinitely-dilute macroion limit. Osmotic pressures are necessarilymeasured at finite concentrations. Once recognized, that necessity

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demands that many features of macroions - concentration, size,flexibility, etc. - be recognized in osmotic compression.

Biotechnology & Bioengineering

633-Pos Board # B502NEW VARIANTS OF FLASH, THE GENETICALLY-ENCODED OPTICAL SENSOR OF MEMBRANEPOTENTIAL.Giovanna Guerrero, Micah S. Siegel, Botond Roska, Eli Loots,Ehud Y. Isacoff; University of California at Berkeley, 279 LifeSciences Addition, Berkeley, CA 94720Genetically-encoded protein based optical reporters allow real timemonitoring of molecular activity. An advantage over fluorescentdyes is that genetically-encoded reporters can be targeted tospecific cell types and subcellular regions. In addition, proteinbased sensors can be modified functionally to achieve differentkinetics and ranges of operation. Here we report progress in thetuning of FlaSh, a previously described GFP-based voltage sensor.By using different GFP versions we have produced sensors withimproved folding at 37°C, as well as distinct excitationwavelengths. Surprisingly, this approach also led to the discoveryof FlaSh variants with dramatically different fluorescence kinetics.Modification of the Shaker moiety of FlaSh also producedreporters with different kinetics. By employing the L366Amutation in Shaker we created a FlaSh with different voltagedependence as well. These modifications on their own and incombination should produce sensors that are specialized fordistinguishing between action potentials or synaptic potentials aswell as reporting voltage changes in different cell types. Inaddition, ratiometric approaches to FlaSh, based on FRET andspectral shifts, have been developed. Insights into the mechanismresponsible for the fluorescent changes of FlaSh are also discussed.

634-Pos Board # B503A NOVEL VOLTAGE-SENSITIVE FLUORESCENT DYEFOR ION CHANNEL AND TRANSPORTER ASSAYSDeborah F. Baxter', Amy F. Garcia', Alejandra Raimondil, MarkE. Jurman', Kimberly K. Flint', Rory Curtis', Martin Kirk2, DieterKlaubert2, Emir Duzic', Dejan Bojanic', Peter S. DiStefanol, YuXiel; 'Millennium Pharmaceuticals, Inc., 75 Sidney Street,Cambridge, MA 02139, 2Molecular Devices, 1131 Orleans Drive,Sunnyvale, CA 94089The study of ion channel-mediated changes in membrane potentialusing the conventional bisoxonol dye, DiBAC4(3), has severallimitations including slow responses and sensitivity to temperature.Here, we report a novel voltage-sensitive fluorescent dye(Molecular Devices, CA) for ion channels and electrogenictransporters. The steady-state and kinetic fluorescence propertiesof the dye are characterized, in comparison with DiBAC4(3), usingboth FLIPR and patch-clamp for a voltage-gated K channel, aligand-gated Ca++ channel, and an electrogenic transporter, GATI.Our experiments reveal that for the voltage-gated K channelexpressed in CHO cells, the new dye is much faster (<10 sec inresponse) than DiBAC4(3). The kinetics of fluorescence changesof the new dye on FLIPR is identical to that seen in whole-cellcurrent clamp. For the ligand-gated Ca" channel, the dye issuperior to both DiBAC4(3) and Fluo-3 in kinetics. We have alsoshown that the dye can be used in assays for GAT1. In GATItransfected HEK293 cells, GABA induces changes of fluorescencein a dose-dependent manor with an EC5o of 2 gtM. 100 9M GABA-induced responses can be blocked by NO-711 with an IC5o of 3FM. This novel membrane potential dye could be a very usefultool for high throughput screening assays for ion channels andelectrogenic transporters.

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635-Pos Board # B504PERFORMANCE TESTING OF THE EVOSCREEN UHTSSYSTEMSabine Schaertl', Anja Wieneckel, Doerte Rolfes', NicoMichaelsen', Philip Gribbon2, Malcolm Wickenden2, MichelleKnight, Martin Daffertshofer'; 'EVOTEC BioSystems AG,Schnackenburgallee 114, Hamburg, 22525 Germany, 2Pfizer Ltd.,Sandwich, Kent CT13 9NJ United KingdomThe EvoScreen Mark2 uHTS system consists of, (I) a compoundreformatting unit ("Mitona") and (II) a screening unit ("Scarina"),comprising assay component dispensing and plate reading.Reformating and assay processes are linked at several stages,through the screening information system (SIS) data base. Theplate layout is common to both processes, and MTP wellcoordinates are designated to the respective Nanocarriers by theSIS. The data base stores a high content of information yieldedfrom the analysis of single molecule fluorescence. Post screening,putative hits are identified using Activity Base 4 (from IDBS). TheMitona and Scarina underwent extended and comprehensivetesting to assess the mechanical handling, fluidic and opticalperformance of the individual instruments and the completeintegrated screening system. Fluidic tests resemble those outlinedfor the Assay Development Stations (see Poster of S. Marose et al.)with which the Mark II shares many similarities. Additional testsaddressed the uHTS capability of the fluidics including qualitycontrolled and high speed dispensing.

636-Pos Board # B505A MICROFABRICATED POTENTIOMETRIC BIOSENSOREmily B Cooper, Juergen Fritz, Nin Loh, Scott R Manalis; MIT,20 Ames Street, Cambridge, MA 02139The miniaturization of biosensors allows the use of small samplevolumes, fast measuring times and the parallelization of sensors. Incombination with a direct detection method which does not requirethe labeling of molecules under detection, these sensors can also beused for point-of-care diagnostics, on-line monitoring or asimplantable sensors. We present a micromachined field-effectsensor with an active sensing area ranging from 5 x 5 pm2 to 100 x100 pm2. The sensor is able to measure changes in surfacepotential that can result from the adsorption of ionic or molecularcharges. The sensing area is integrated on a silicon cantilever andhas been shown to measure pH changes in channels of amicrofluidic network (1). We are currently using this device forinvestigating immunospecific cell detection and DNAhybridization.(1) S.R. Manalis et al.: Microvolume field-effect pH sensor forthe scanning probe microscope, Appl. Phys. Lett 76 (2000) 1072.

637-Pos Board # B506A RESISTIVE SENSING DEVICE FOR BIOLOGICALSOLUTIONSOmar A. Saleh, Lydia L. Sohn; Princeton University, Jadwin Hall,Washington Rd., Princeton, NJ 08544We present a microfabricated device able to measure the size andconcentration of sub-micron particles in solution. The deviceconsists of two reservoirs connected by a small aperture, and filledwith particle-laden solution. Particles passing through the aperturedisplace conducting fluid, raising the electrical resistance betweenthe reservoirs. The size and frequency of the resistance changescan be directly related to the particle diameter and concentrationrespectively. We have successfully measured particles withdiameters down to 190 nm in this manner. We discuss theapplicability of this device towards measurements of DNA andproteins, along with the possibilities of chemical functionalizationthrough surface modification of the aperture.

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638-Pos Board # B507CHARACTERIZATION OF A LIVER TISSUE BASEDBIOSENSORAdam T Capitano, Mark J Powers, Anand Sivaraman, Peter T So,Linda G. Griffith; Massachusetts Institute of Technology, 77 mainst room 379 building 56, cambridge, Ma 02141Since the liver is the primary organ in the body for processingxenobiotic species, a liver tissue based biosensor potentially offersmany advantages for toxin detection. However, growth of livertissue is challenging since liver loses hepatic function on the orderof days using standard cell culture techniques. To circumvent thisproblem, we have developed a perfusion bioreactor which providesa stable, 3-dimmensional structure as well as physiological flowconditions for nutrient and gas exchange. Two-photon microscopyhas been used to characterize the viability of hepatocytes culturedwithin this reactor. Hepatic functionality as measured bycytochrome p450 lAl production has been maintained at constantlevels as long as two weeks post isolation. Production of thisenzyme has been used as a marker for toxin detection. Bothcalcium ionophore as a fast acting agent and aflatoxin as a slowacting agent have been detected demonstrating that this sensingplatform can be used for a wide range toxins.

639-Pos Board # B508CAPACITANCE CYTOMETRY: MEASURINGBIOLOGICAL CELLS ONE-BY-ONELydia L. Sohn, Omar A Saleh, Geoffrey R. Facer, Andrew J.Beavis, Richard S. Allan, Daniel A. Notterman; PrincetonUniversity, Jadwin Hall, Princeton, NJ 08544Measuring the DNA content of eukaryotic cells is a fundamentaltask in biology and medicine. We have recently developed aninnovative new technique-"capacitance cytometry"- whichallows one to quantify-all on an integrated microfluidic chip-theDNA content of single eukaryotic cells. This is accomplished bythe measuring the change in capacitance as single cells pass acrossa 1 kHz electric field. By measuring a variety of cells fromdifferent organisms, ranging from yeast to mammals, we havediscovered a species-independent linear relationship between theDNA content of eukaryotic cells and the associated change incapacitance. In addition, we have demonstrated that capacitancecytometry allows one to examine the cell-cycle kinetics ofpopulations of cells. Consequently, this technique may in thefuture serve as a medical diagnostic tool whose low-detection limitof just one cell can identify the presence of aneuploidy in a very

small quantity of tissue, and without special processing.

640-Pos Board # B509FUNCTIONALIZED LIPOSOMES FOR STABILIZATIONAND PROTEOLYSIS PROTECTION OF ENZYMESMathias Winterhalterl, Wolfgang Meier2, Didier Fournier3;'CNRS, 205, rte de Narbonne, Toulouse,F-31077 France, 2Phys.Chemie, Basel, CH-4056 Switzerland, 3UMR 5068, 118, rte deNarbonne, TOULOUSE, F-31062 FranceThe stability of proteins in solution is limited by degradationthrough proteases or in diluted samples by self-denaturation.Unfortunately many biotechnical applications of enzymes requireenvironments rich in proteolytic enzymes and concentrationsfavoring destabilization resulting in a subsequent rapid loss inactivity. To overcome this limit, we propose a novel technique tocontrol the permeability of the liposomes using naturally occuringmembrane channels with selected features. As an example we use

OmpF, a channel from the outer cell wall of Escherichia Coli. Ithas the advantage to be extremely stable and now available in largequantities which opens new technological applications. Wedemonstrate that insect acetylcholinesterase encapsulated intofunctionalized lipid nanocontainers is fully active, protectedagainst protease and stable even in very diluted samples

641-Pos Board# B510MICROTEXTURED SURFACES PRODUCE BETTERCARDIAC MYOCYTE CULTURESDelara Motlagh, Samuel Y Boateng, George Nijmeh, JamesReinhardt, Brenda Russell; University of Ilinois @ Chicago, 835South Wolcott Ave (M/C 901), Chicago, IL 60612The objective is to alter surface topography in order to influencecell shape and study the structure/function relationship in thecardiomyocyte. Microtextured silicone membranes are made usingphotolithography and microfabrication techniques and laminincoated. Primary rat neonatal cells are plated on flat, micro-pegged(5 or 10tm high), or parallel micro-grooved (5Sim deep)membranes. Cells attach more often to an actual peg (90±1%;n=3)than to a virtual peg (15±3%;p<0.0001) by a cell crawling process.Myofibril height is thicker for cells grown on pegged than flatmembranes (n=5,p=0.0004). Cells are more aligned on grooved(72±1%;p=0.001) than on pegged(0.5±6%) or flat (3±1%;n=5)membranes. Actin stress cables are shorter on pegged(2.5Mm±1.6%) as than flat (30Om±2%) or grooved (3814m±2%)membranes. Fibroblast hyperplasia is significantly reduced onpegged membranes. Pegs survive pulsatile stretching (Bioflex3000, lHz, 48 h) in the physiological range (10% strain) and cellattachment is not significantly changed. This new culture systemproduce aligned cells that may generate anisotropic force as in vivowith increased myofibrillar mass in the transverse direction thatmay permit greater stress production. Furthermore, proliferation offibroblast may be reduced. HL62426

642-Pos Board # B511QUANTITATIVE ANALYSIS OF HEPATOCYTESPREADING ON ADHESIVE SUBSTRATES.Arpita Upadhyaya, L. Mahadevan; Massachusetts Institute ofTechnology, 77 Mass. Ave., Cambridge, MA 02139When a spheroidal aggregate of cells is placed on an adhesivesubstrate, it spreads on the surface. We have quantitativelyobserved the spreading of hepatocyte spheroids on collagen coatedsubstrates. We find that the initial rate of spreading is linear with agradual slowing down in the final stages. We studied the effect ofspheroid sizes and collagen concentration on the spreading rate.We can explain these results by considering a balance between theadhesion and elastic energies of the tissue. The tendency of thespheroid to maximize its contact area with the substrate in order toincrease the number of adhesive bonds is balanced by its effectiveelasticity which resists deformation. The ratio between the twoenergies provides a length scale which determines the equilibriumshape of spheroids depending on their initial size. The dynamics ofthis process depends on how the cells move relative to each otherand the substrate.

643-Pos Board # B512CONTROL OF CELL ADHESION AND GROWTH WITHMEMBRANE MICRO-ARRAYSJay T. Groves, Lara K. Mahal, Carolyn R. Bertozzi; UC Berkeley,Calvin 206, UC Berkeley, Berkeley, CA 94720In this work phospholipid bilayers are employed as biomimeticcoating materials to modulate the adhesion and growth of cells onsolid substrates. A variety of lipid compositions and chargedensities are examined. Culturing cells on these supportedmembranes reveal that fluidlipid bilayers generally block celladhesion with a notable exception provided by membranescontaining phosphatidylserine, which strongly promote adhesionand growth. This dichotomy is utilized with micropatternedmembrane technology to selectively direct cell growth to specifiedregions on a substrate. Lipid composition in micropattemedmembrane arrays is demonstrated to be a simple and effectivemeans of patterning cell growth on surfaces.

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644-Pos Board # B513APPLICATION OF SILICONE MICROCHANNEL ARRAYIN AUTOMATED SINGLE RED BLOOD CELL ANALYSISJure Derganc', Michael G. Frank', Sean C. Gifford', TatsuroYoshida', Christopher Gabel', Robert H. Austin2, Mark W.Bitensky'; 'Boston University, 2Princeton UniversityManual measurements of individual red blood cell area andvolume are technically demanding and much too slow for thepurpose of examining a statistically significant number of cells.We have therefore developed a device, which utilizes disposablearrays of many identical microchannels in transparent siliconerubber, for accurate single red cell measurements. The cells can beaspirated into the microchannels much like they are in themicropipette. Since there are hundreds of identical microchannelsin each array, many cells can be individually and preciselyanalyzed in a rapid automated process. Our array uses wedge-shaped channels which are designed to trap the red cells. At thepoint of arrest, the cells are pressurized and the membrane enclosesits volume without folds. The positions of the trapped cells arereadily determined and, since the geometry of the channels is well-defined, each cell's position can be translated directly intomeasurements of cell volume and area. The trapped cells do notadhere to the lubricated microchannels and can be observed in realtime as they respond to chemical or osmotic changes in theperfusing buffer. Moreover, the individual cell hemoglobinconcentration can be measured and cell surface markers identifiedwithin the channels and thus correlated with cell area and volume.

645-Pos Board # B514ERYTHROCYTE MEDIATED LEUKOCYTE-ENDOTHELIAL WALL INTERACTIONSCristiano Migliorini, Jin Yuan, Rakesh K. Jain, Lance L. Munn;Massachusetts General Hospital/ Harvard medical school, 100Blossom st.- Cox 7, Boston, MA 02114Because of the complexity of the problem, efforts attempting torelate the biochemical and rheological properties of red blood cells(RBCs) with leukocyte adhesion in vivo have been thus far limited.Recent work from our laboratory has shown that circulating RBCsplay a critical role in mediating leukocyte adhesion to endothelialwalls both in vitro and in vivo. In fact, in the absence of RBCsleukocyte adhesion is a highly improbable event. Therefore, abiophysical analysis of the problem is needed to incorporate themechanical influences of the blood flow into existing models ofleukocyte adhesion. RBCs in the suspending medium affectleukocyte adhesion by changing the leukocyte distribution in thevessel, by increasing the wall collision frequency and by changingthe forces acting on adhering leukocytes. We have analyzed thesefundamental mechanisms independently using in vitro modelswhere the rheology of RBC suspensions can be controlled. Resultsfrom measurements of leukocyte distribution, rolling, adhesion anddeformation in cylindrical and rectangular flow chambers aresynthesized in a mathematical model accounting for the multiplephenomena that are thought to play major roles in the process ofcell adhesion in vivo. The results indicate that the rheology ofblood and the visco-elastic properties of the particles in the flow(including RBC aggregation and deformation) are criticalparameters in the delivery and adhesion of leukocytes toendothelial walls.

646-Pos Board # B515DIELECTRIC PROPERTIES OF MAMMALIAN LIVERTISSUES EXPOSED TO X-RAYS.Christopher Bassey; Western Kentucky University, I Big RedWay, Bowling Green, Kentucky 42101The relative permittivity and alternating current conductivity ofexcised bovine liver tissues have been studied before and afterexposure to 110 mR x-rays. Measurements were made in thefrequency range from 1 to 50 MHz by means of a resonance

644-648

technique, at an average temperature of 26.5 degrees. For tissuesnot exposed to radiation, results showed a decrease in relativepermittivity and an increase in conductivity with frequency, inagreement with previous studies. However, for tissues exposed to110 mR x-rays, higher values of relative permittivity andconductivity were found. The difference in relative permittivitybetween the exposed and non-exposed tissues was wellpronounced below 10 MHz and it decreased with frequency.Higher values of conductivity were also noted for the exposedtissues and this was observed over the entire range of frequencies.Other dielectric parameters such as loss factor, Cole-Cole spreadindex and relaxation frequency also showed higher values. Theincrease in dielectric properties is attributed to the presence of ionsin the tissues after exposure to radiation. The mechanism isexplained by the accumulation of charges at the regions ofdiscontinuity in the tissue due to its inhomogeneous nature. This isthe Maxwell-Wagner polarization mechanism. This technique canbe used to monitor radiation contamination of meat samples.

647-Pos Board # B516DETECTING ACTIVE PEPTIDES AND PROTEINS INTETHERED LIPID BILAYERSJohn T. Elliott, Vitalii Silin, John T. Woodward, David J.Vanderah, Curtis W. Meuse, John J. Kasianowicz, Anne L. Plant;National Institute of Standards and Technology, 100 Bureau Drive,Gaithersburg, MD 20899We have fabricated a tethered phospholipid bilayer sensor platformfor detecting the function of spontaneously insertingtransmembrane peptides and proteins. First, a mixed -mercaptoethanol/1-thia-hexa(ethyleneoxy)-octadecanoatemonolayer is assembled on a planar gold surface. Theethyleneoxide portion of the amphipathic anchor is designed toprovide a thin aqueous layer between the solid support and thebilayer membrane that will accommodate small extramembranousregions of transmembrane proteins. Second, 1-palmitoyl-2-oleoyl-phosphatidylcholine is applied to the mixed monolayer in anethanol solution and diluted with aqueous buffer. Impedanceanalysis suggests the formation of a low dielectric bilayer structure(Cm-O.9 pF/cm2) over a hydrated aqueous layer (-9 gF/cm2). Thebilayer is highly insulating and defect free as determined by ACimpedance analysis in the presence of the soluble FeCN63'4 redoxcouple. Addition of the peptide toxin, melittin, results in theformation of defects that allow FeCN63/4- ions access to the goldelectrode. We are now using simultaneous surface plasmonresonance and impedance measurements to further characterize thephysical structure of the tethered membrane and the defects thatare formed by melittin. We are also extending these studies todetect the presence of the spontaneously inserting pore formingprotein, a-hemolysin (aH). Reconstitution of axH into a biosensorplatform may allow us to detect DNA oligomers that alter theconducting properties of the aH pore.

648-Pos Board # B517HORIZONTAL BILAYER PAINTED ON A SYSIO2 CHIPRigo Pantoja', Rikard Blunck2, Daniel M. Sigg2, James R. Heath',Francisco Bezanilla2, ; 'UCLA Dept. of Chemistry andBiochemistry, Los Angeles, CA 90095, 2UCLA Depts. ofPhysiology and Anesthesiology, Los Angeles, CA 90095Reconstituted ion channels in painted bilayers serve as a welldefined model system for optical and electrical investigations of

their structure-function relationships.Horizontically oriented bilayers withelectrolyte access to both sides wereobtained by microfabricating a silicon

-5- dt $"$bJpartition, that was matched to the

configuration of a microscope with a highnumerical aperture objective. Standard lithographic techniques and

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anisotropic deep-reactive ion etching were used to micromachinepores of diameters 50 to 100 gm. Silanization of the SiO2 surfaceproduced the surface hydrophobic properties required to paintphospholipids on the partitions. The cylindrical structure of thepore in the partition and the surface treatment resulted in extremelyrobust bilayers, which stayed constant after reconstitution ofMaxiK channels.Supported by Keck Foundation, NSF and NIH GM30376

649-Pos Board # B518REOREENTATION OF THE a-HELICAL SYNTHIETICPEPTIDE ZNPPIXBBC16 IN A LANGMUIRMONOLAYERAndrey Tronin1, Joseph Strzalka', Xiaoxi Chen2, BenjaminOcko3, P. Leslie Dutton', J. Kent Blasie'; 'University ofPennsylvania, Philadelphia, Pennsylvania 19104, 2HarvardUniversity, 3Brookhaven National LaboratoryThe vectorial orientation of the peptide ZnPPIXBBC16 in aLangmuir monolayer was studied by means of X-ray reflectivityand polarized epifluorescence. ZnPPIXBBC16 consists of two a-helical 31-mers dimerized via a disulfide bridge between two N-terminal cysteines (a-S-S-a), so that two bis-his metalloporphyrinbinding sites at positions 10 and 24 are formed.Zn(Il)Protoporphyrin IX was bound to the position 24. To increasepeptide amphiphilicity, a palmitoyl (C16) chain was bonded toeach N-terminal cysteine. X-ray reflectivity measurements make itpossible to infer the orientation of the a-helices by determining theelectron density profile of the monolayer. Polarizedepifluorescence provides the orientation distribution of theporphyrin with respect to the monolayer plane. Both x-ray andfluorescence measurements show that at low surface pressure (it =5 mN/m) the peptide a-helices are oriented parallel to the watersurface with a narrow orientational distribution of the porphyrin.At high pressures (i > 30 mN/m), the peptide a-helices areperpendicular to the water surface, and the distribution is wider. Atthe intermediate pressures the peptide a-helices in the monolayerundergo a transition from the parallel to perpendicular orientationwith almost a complete loss of the orientational order of theporphyrin.

650-Pos Board # B519EVALUATING A LIPIDIC CUBIC PHASE FOR USE INENVIRONMENTAL REMEDIATION: WASTEWATERTREATMENT WITH MYVEROL.Jeffrey Clogston, Martin Caffrey; The Ohio State University, 176W. 19th Ave, Columbus, OH 43210Myverol 18-99K is a commercial fat rich in monoacylglycerols,monoolein in particular. When fully hydrated, the cubic (Pn3m)phase forms spontaneously and is stable over a wide temperaturerange (Clogston et al., 2000. Chem. Phys. Lipids. In press). Thecubic phase is made up of a pair of interpenetrating but non-contacting aqueous channels separated by a single, highly curvedcontinuous lipid bilayer with an estimated polar/apolar interfacialarea of 485 m/g lipid. Because Myverol is a relativelyinexpensive and biocompatible material, we are exploring thepossibility of using the "sponge-like" properties of a Myverol-based cubic phase in wastewater remediation. Accordingly, theuptake of phenol, a wastewater contaminant of note, into pre-formed bulk cubic phase prepared from Myverol has beenquantified. This is compared to uptake by "neat" or pure Myverol,by a colloidal dispersion of the Myverol-based cubic phasereferred to as cubosomes and by activated carbon. Cubosomes arestabilized by the biocompatible detergent Pluronic F-127. Thephase behavior of the Myverol/Pluronic/water system at threetemperatures, characterized by x-ray diffraction, is reported.Support: NIH (DK36849, DK46295, GM56969), NSF (DBI9981990), The Ohio State University Interdisciplinary Program.O

651-Pos Board # B520MOLECULAR DYNAMICS STUDIES OF PROTEININTERACTION WITH AU CRYSTAL SURFACESRosemary I. Braun', K. Schulten', D. Heidel2, M. Sarikaya2;'University of Illinois, 405 N Mathews, Urbana, IL 61801,2University of Washington, SeattleBiological control of inorganic crystal morphology is essential tohard tissue growth and is useful for materials engineeringapplications. Gold binding proteins (GBPs) have been geneticallyengineered by Sarikaya and coworkers. It was shown that in thepresence of GBP, gold formed large, flat hexagonal crystalsdisplaying the (111) surface. No such crystals were seen to form inthe presence of control proteins which do not bind to gold. It ishypothesized that GBP binds preferentially to the (111) Ausurface, and that the covering of the (111) face by the bound GBPplays a role in the mechanism by which GBP alters crystalmorphology. It is not readily apparent how the GBP adheres togold, nor why the (111) surface would be preferred to, eg, the moresparsely populated (211) face. Both chemisorption (via GBP'smethionine sulfurs) and physisorption (via polar side-chains) couldplay a role in the binding. We have predicted structures for thethree GBP sequences available; two are seen to have repeatingmotifs which may be conducive to binding to a periodic surface.To investigate the role of physisorption, we have conducted 5nsMD of the protein-gold system for both the (Il1) and (211) planes.For both cases, the protein is stable after 500ps, and the polar side-chains are seen to be in contact with the Au surfaces. Thecorrugations in the (211) plane are found to be more accessible towater. We also present results of steered molecular dynamics, inwhich the GBP is pulled across the Au surfaces.

652-Pos Board # B521ELECTRONIC CHARACTERIZATION OF BIOLOGICALFLUID SAMPLES: 40 HZ TO 30 GHZGeoffrey R Facer, Daniel A Notterman, Lydia L Sohn; PrincetonUniversity, Physics Department, Jadwin Hall, Princeton, NJ 08544We report developing coplanar waveguide (CPW) devices whichcan electronically characterize biological samples withinmicrofluidic channels. Purely electronic analysis is a rapid andsensitive probe of macromolecular solutions and cell suspensions,which circumvents some of the limitations and complexity ofoptical measurements (for example, the need for fluorophoretagging, or photobleaching effects). Our CPWs function in twomodes: broadband swept-frequency measurements (dielectricspectroscopy), and fixed-frequency acquisition to probe time-dependent phenomona or passing individual cells. Dielectricspectroscopy data obtained with these devices covers an extremelybroad frequency range: from 40 Hz to 30 GHz. We demonstratethe ability to distinguish between different proteins, and betweenlive bacteria and inert colloidal particles. At a fixed frequency, weare able to monitor time-dependent effects over time scales frommilliseconds to hours. Coupling to the fluid sample is capacitive,avoiding many of the problems associated with ionic screening atthe electrodes. Also, capacitive coupling removes the need forsurface functionalization. These two advantages greatly increasethe potential applications for electronic characterizationtechniques.

653-Pos Board # B522ELECTROTRANSFORMATION OF SKELETAL MUSCLEFIBERS WITH HIGH VOLTAGE RADIO FREQUENCYMODULATED SHORT PULSESMichael Tyurin, Martin F. Schneider; Dept. of Biochem. & Mol.Biol., Univ. ofMD School of Medicine, Baltimore, MD 21201Single unipolar square pulses (12.5 kV/cm, 1 ms duration)modulated at a frequency of 1MHz were applied to 100 - 250mouse isolated FDB muscle fibers suspended in 50 ,l of HEPESbuffered MEM with 10 tg of either pHCMV--0-Gal or pAdlox

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(GFP).plasmid DNA in a 0.5 ml Eppendorf tube on ice. Fiberswere then plated on larninin-coated coverslips and maintained inculture. Electrotransformation (ET) yields of 12 - 15 % at fiberviability of about 50% were obtained. ET of muscle fibers in the50-60 gl volume required a momentary power of about 4 kW persample. A model describing V. formation at a small restricted areaof fiber surface is proposed. While I-Gal expression could bedetected after overnight culture, monitoring of GFP expressed byliving fibers showed increased transformation yields to 72 h inculture. Replacement of PBS in culture medium with 7 Wg/ml ofPrednisolon maintained fibers in good condition for 5 days afterelectric treatment. For GFP expressed after 40 h in culture,transformants maintained morphology of viable fibers for up to112 h of monitoring. This is the first report on efficient ET ofmature skeletal muscle fibers, and the first report on efficient ET ofeukaryotic cells with high voltage short pulses. Supported by NIHROI-NS33578 and T32-AR07592.

654-Pos Board # B523SIMULTANEOUS MR IMAGING AND REGIONALHYPERTHERMIA: FIRST CLINICAL RESULTS.Volker F. Kurzel, M. Peller2, A. Baur2, S. Abdel-Rahman', M.Santl', M. Reiser2, R. Issels'; IMed. Klinik m, KlinikumGrolBhadern, Universitait Muinchen, Marchioninistr. 15, Muinchen,81377 Germany, 2Klinikum Grol3hadern, Universitait MtUnchen,Marchioninistr. 15, Muinchen, 81377 GermanyRegional hyperthermia in combination with chemotherapy or/andradiotherapy has proven to be an effective treatment concept forlocally advanced deep-seated tumors. Routinely, one-dimensionaltemperature measurements are performed along an invasivelyimplanted catheter. The purpose of this project is the improvementof the thermometry using 3-dimensional MR imaging duringregional hyperthermia. A new MR-hyperthermia hybrid-systemwas installed and tested in phantoms and under clinical conditionsin patients. The tests included operation with therapeutichyperthermia power levels during imaging. We achievedsimultaneous MR-imaging and Tl -relaxation time determination at0.2 Tesla during RF heating with all types of pulse sequences. TIrelaxation time changes were detectable during the hyperthermictreatment. This could constitute a basis for non-invasive MR-thermometry. We used subtraction of TI parameter maps acquiredbefore and during heating to visualize the changes in TI. Thesefirst clinical applications of an MR-hyperthermia hybrid-systemare an important step towards improved regional hyperthermia oflocally advanced deep-seated tumors. The patterns of Ti relaxationchanges during hyperthermia treatment may prove to be useful forspatially resolved thermometry and thus help improve thehyperthermia therapy.

Calorimetry

655-Pos Board # B524THERMAL DENATURATION STUDIES OF THEISOFORMS OF HUMAN APOLIPOPROTEIN E.Prathima S Acharyal, Mark Segall2, Mohammed Zaiou2, JulieMorrow3, Karl Weisgraber3, Michael C Phillips2, Sissel Lund-Katz2, Julian Snow'; 'University of the Sciences in Philadelphia,600 South 43rd Street, Philadelphia, PA 19104, 2Children'sHospital of Philadelphia, Philadelphia, PA , 3Gladstone Institue ofCardiovascular Research, University of California, San Francisco,CAAs a component of serum lipoprotein particles, apo E plays a keyrole in lipid transport. Human apo E exists in three commonisoforms (E2, E3 and E4), characterized by Cys/Arg interchangesat positions 112 and 158 and this polymorphism is associated withdyslipidemia and neurological defects. To further understand themolecular basis for these effects, DSC has been used to study theconformational changes that occur upon thermal unfolding of theseisoforms and their N- and C-terminal fragments. We havemeasured differences in the thermal stabilities of the isoforms (E2

> E3 > E4) and have been able to quantify these differences interms of free energies of unfolding. The 22-kDa fragments unfoldat lower temperatures (Tm = 59, 55 and 520C for apo E2, E3 & E4,respectively) and lower enthalpies (AH = 61, 63 and 69 kcalUmolefor apo E2, E3 and E4, respectively) than their full-lengthcounterparts. We have detected the presence of unfoldingintermediates (both by DSC and CD) during the thermaldenaturation of 22-kDa fragments of apo E3 and E4 but not of E2.The thermal unfolding of the full-length proteins is stronglyinfluenced by their state of aggregation, with self-associationcausing enhanced thermal stability. Further lipid-free apo E3 andE4 (but not E2) seem to be partially unfolded at physiologicaltemperatures.

Single Molecule Biophysics

655.01-Pos Board # B525A NEW STRATEGY FOR SEQUENCING INDIVIDUALMOLECULES OF DNAJonas Korlach, Michael Levene, Stephen W Turner, Dan RLarson, Mathieu Foquet, Harold G Craighead, Watt W Webb;Cornell University, Clark Hall, Ithaca, NY 14853A new optical method for determining the base pair sequence of asingle molecule of DNA is described. In its principle, the activityof a single molecule of DNA polymerase on a template nucleicacid is followed in real time. The sequence is deduced byidentifying which base is incorporated into the growingcomplementary strand of the target nucleic acid at each step in thepolymerization reaction. The technical challenges consist in thedevelopment of suitable enzymatic systems and in the opticalrecognition of individual sequential base additions. The approachwill utilize novel fluorescently labeled nucleotide analogs as

substrate molecules for the polymerase. Temporal resolution ofsequential nucleotide additions is made possible by time-resolvedphoton burst analysis. Several techniques for confinement of theexcitation volume far below the diffraction limit are presented,such as submicrometer sized fluid channels and near-field aperturearrays. These nanostructured devices permit an increase insubstrate concentrations by about three orders of magnitude, up tothe micromolar range (required for the enzymatic system understudy), while still allowing sequential single molecule recordingsand analysis.

655.02-Pos Board # B526SINGLE MOLECULE CHARACTERIZATION OFPOLYNUCLEOTI]DES USING A TRANS-MEMBRANECHANNELAmit Mellerl, Lucas Nivon', Daniel Branton2; 'The RowlandInstitute for Science, 100 Edwin H. Land Blvd., Cambridge, MA02142, 2Harvard University, 16 Divinity Ave., Cambridge, MA02138The trans-membrane channel, a-hemolysin, can be used to localizeand detect single DNA and RNA molecules on a microsecondtimescale. The individual macromolecules are driven into thechannel and across the membrane by a voltage bias. Detectionrelies on probing the reduction in the ionic current as the moleculestranslocate through the channel. The ionic current is modulated byboth the local cross-section and structure of the probed molecules.For each translocation event we record the average current leveland duration. We find that the current level and durationdistributions contain information about the type ofDNA moleculesand their local structure. Different types of DNA homopolymers,such as poly(deoxyadenine) and poly(deoxycytosine) in a randommixture, are rapidly discriminated based on this approach. Thedistributions obtained for polypurines differ from those ofpolypyrimidines, a fact that we attribute to the differences in theirsecondary structures. In our recent experiments we study how the

passage duration depends on temperature, polymer length and thedriving potential. Our results suggest that a combination of

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entropic and drag contributions dominate the polymer motion. Weexpect that these experiments will refine and extend existingmodels of driven polymer translocation kinetics.

655.03-Pos Board # B527MOLECULAR MOTIONS OF A LONG DNA CONFINED INA SMALL SPACEMasanori Ueda, Noritada Kaji, Yoshinobu Baba; The Universityof Tokushima (JST), 1-78 Shomachi, Tokushima, Tokushima 770-8505 JapanWe studied electrophoretic-behaviors of a long DNA molecule(48.5kbp, 166kbp, and lMbp) confined in a small space. DNA wasstained with fluorescence dye, and incubated in linear polymersolutions or gel systems etc. The solution including DNA wasplaced on a miniaturized electrophoretic cell on a fluorescencemicroscope. We applied AC electric fields (sinusoidal and pulsedfields) to the solution, observing the molecular motions of theindividual DNA.DNA shows several types of motion, depending on the externalfield's frequency, voltage, and the obstacle's concentration.Especially, under the electric field around 10 Hz and 200 V/cm,DNA is stretched out almost fully because of the entanglementbetween DNA and obstacles. This stretching is observed with awide range ofDNA sizes (48.5kbp - lMbp) and gel concentrations(agarose, 0.5% - 2%). In order to explain the DNA stretching, wepropose a simple mechanism by a tube model. We will taLk aboutthe possible application of the DNA stretching to biologicalmethods.

655.04-Pos Board # B528iMAGING OF SINGLE MRNA MOLECULES MOVINGWITHIIN A NUCLEUS OF LIVING CELLSHisashi Tadakumal, Toshiharu Shibuya2, Yo lshihama',Junichirou Atoh', Yasuaki Habara2, Tokio Tani2, Takashi Funatsu';'Waseda University, 2Kyushu University, JapanMessenger RNA is transcribed and processed in the nucleus andtransported to the cytoplasm. In order to clarify the molecularmechanism of these processes, we have developed video-rateconfocal microscopy that enabled us to visualize singlefluorescence molecules. Movement of individual mRNAmolecules within a living cell was visualized by this microscopy todistinguish whether mRNA is directed by active transport or

moving around by diffusion. For this purpose, Cy3-labeledtruncated human 3-globin mRNA molecules were prepared in vitroand micro-injected into the nucleus of Xenopus A6 cell. Cy3-labeled mRNA molecules could be observed except nucleoli,indicating that mRNA could access most of the space, excludingnucleoli, in the nucleus. Some population of mRNA movedaround in diffusion, and some remained stationary. Statisticalanalysis of individual mRNA trajectories revealed that about 50%of mRNA is moving at the diffusion constant of 0.4 [pm2/s]. Thisvalue is about 1/25 of that in solution ( 9 [ptm2/s]). Our resultssupport the mechanism that mRNA reach nuclear pores bydiffusion process.

655.05-Pos Board # B529VISUALIZATION AND TRACKING OF SINGLE UlSNRNP WITHIN THE CELL NUCLEUSThorsten Kues1, Achim Dickmanns2, Reiner Petersl, ReinhardLUlhrmann2, Ulrich Kubitscheckl; lWestfllische Wilhelms-Universitat Mtinster, Mtlnster, D-48149 Germany, 2Max-Planck-Institute for Biophysical Chemistry, Gdttingen, D-37077 GermanyThe U small nuclear ribonucleoproteins (snRNPs) are componentsof the splicing complexes that remove introns from pre-mRNA.Investigating the localisation within the nucleus by confocal laserscanning microsocpy U snRNPs are generally found diffuselydistributed, but additionally are also associated with severaldistinct nuclear substructures, including interchromatin granules,

perichromatin fibrils, and coiled bodies. We have studied theintranuclear movements of wildtype Ul snRNP particles on asingle particle level using sophisticated high speed and highsensitivity laser microscopic methods. Ul snRNP particles werefluorescently labeled with Alexa 488, and imported into nuclei ofdigitonin semi-permeabilized 3T3 mouse cells in a signaldependent manner. Images taken on a millisecond time scale werefurther processed using especially developed image analysis tools.The data revealed that the observed single Ul snRNPs were slowlymoving (D < 1Am2/s) - but were never completely immobile -when residing in selected nuclear regions that probably correspondto the above cited intranuclear structures, in the followingdesignated as speckles. Within speckles the particles could belocalized with an accuracy of < 20 nm. Speckles hosted UlsnRNPs only for a limited time span which extended up to 1 s. Adetailed motion analysis of Ul snRNPs observed within speckleswill be presented. Outside speckles Ul snRNPs moved fast, i.e.with diffusion constants> 10 Mnm2/s.

655.06-Pos Board # B530IN VIVO PHARMACOLOGY DOWN TO THE SINGLEMOLECULE LEVELGerhard J Schuetz1, Guenter Freudenthaler', Jan Hesse', BerntPragl2, Hans-Guenter Knaus2, Hansgeorg Schindler'; 'Universityof Linz, Altenbergerstr.69, Linz, A-4040, 'University of InnsbruckRecent applications of ultra-sensitive fluorescence microscopy toliving cells allowed, for the first time, the direct observation ofcellular processes on the single molecule level. The generalapproach of observing individual copies of a protein expressed onthe plasma membrane of a cell by using fluorescence labeledligands allows for an in vivo determination of its pharmacologicalprofile on a single cell level. We show here a characterization ofthe binding properties of fluorescence labeled Hongotoxin to thepotassium channel Kvl.3 in human T-lymphocytes in terms of thedissociation constant, the off-rate, and by competitive inhibitionwith unlabeled toxin. In addition, the low expression of the Kvl.3on Jurkat cells allows to resolve single fluorescence labeled ionchannels as diffraction limited peaks. We utilized the highpositional accuracy of single molecule microscopy of 50nm todetermine a full 3D map of individual channel proteins on a singlecell.Supported by the Austrian Ministry of Science, GZ 200.025/3.

655.07-Pos Board # B531HIGH THROUGHPUT SINGLE DYE TRACINGJan Hesse, Gerhard J. Schuetz, Hansgeorg Schindler; Universityof Linz, Altenbergerstr.69, Linz, A-4040New emerging perspectives in the field of functional genomics arebased on the knowledge of the sequence of the human genome. Inthis post-genomic age, the main focus is shifting from puresequence analysis to the study of gene transcription, detected viamRNA, and of the finally expressed proteom. Fast screening oflarge amounts of cells in different microenvironments will be thebasic requirement for the beginning era of proteomics. We presenthere a technique which allows for screening of up to 106 cells in40min with single molecule sensitivity. Using a high accuracystage, the samples can be repeatedly scanned, which allowssequential cell treatment and exact correlation to previous results,e.g. after addition of ligands, hormones. This opens newperspectives in the investigation of protein expression, distributionand colocalization. The presented method is an enhancement ofconventional single dye tracing towards high throughput screeningsystems. It utilizes an epimicroscope equipped with a xy-stage inconnection with an ultra sensitive CCD-camera capable of singlemolecule imaging. The CCD readout rate of lMHz allows forscanning sample dimensions up to 1cm2within 40min.

(Patent PCT/AT 99/00257, international registration 28/10/99)

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655.08-Pos Board # B532

IDENTIFICATION OF SINGLE MOLECULES BYANALYSIS OF THE INTENSITY DISTRIBUTION.

giuseppe chirico', Francesca olivini', Sabrina beretta', albertodiaspro2; 'Unversity of Milan, piazza delle scienze 3, Milano, italy20126, 2university of Genoa

We have studied single molecules and molecular aggregates ofrhodamine 6G and labeled proteins on glass substrates by detectingthe two-photon excited fluorescence. The fluorescence intensity ofthe spots occurs at definite values that are multiples of a referencesignal and extrapolate to the background level measured on theimages. These properties and the chaotic time behavior of thefluorescence displayed by the dimmest spots on the images,suggests that these spots arise from single rhodamines. Thediscrete character of the intensity distribution can therefore be usedas a simple, quantitative tool to discriminate between singlemolecules and molecular aggregates on single snapshots. Thesestudies are extended to betalactoglobulin labeled with Alexa-532adsorbed on glasses, in gels and chemically bound to silanizedglass surfaces.

655.09-Pos Board # B533FLOURESCENCE CORRELATION SPECTROSCOPY INZEPTOLITER VOLUMESDaniel R. Larson, Michael Levene, Steve W. Turner, Harold G.Craighead, Watt W. Webb; Cornell University, Clark Hall, CornellUniversity, Ithaca, NY 14850

Fluorescence correlation spectroscopy (FCS) is a technique formeasuring non-equilibrium quantities such as diffusioncoefficients, rate constants, and oligomerization rates forfluorescent species in solution. These single molecule studies are

usually performed at nanomolar or lower concentrations.However, it is often desirable to obtain information on the singlemolecule level at higher concentrations, for example in the studyof enzymatic processes at physiological concentrations of ligand.We have developed an analytical technique that is an extension oftraditional near-field microscopy techniques and is based on theenhanced electric fields present near acicular metal objects ofnanometer radii of curvature. We form silver wedges on a siliconsubstrate through evaporation and subsequent photolithographicetching. The radius of curvature produced at the tip of the wedgeis on the order of 15 nm. Multiphoton excitation is used to excitedyes that diffuse near the tip, and the size of the enhanced focalvolume is determined by the average residence time of themolecule in the volume. We estimate that the focal volume isapproximately 5 orders of magnitude smaller than a diffractionlimited focal volume. This technique enables studies of singlemolecules present at micromolar concentrations.

655.1-Pos Board # B534NEAR-FIELD APERTURES FOR REDUCED-VOLUMEFLUORESCENCE CORRELATION SPECTROSCOPY ANDSINGLE MOLECULE STUDIES.Michael Levene, Jonas Korlach, Stephen Turner, HaroldCraighead, Watt W. Webb; Cornell Univeristy, Clark Hall, Rm.212, Ithaca, NY 14853

Fluorescence correlation spectroscopy (FCS) is a powerfultechnique for elucidating concentration, diffusion and reactiondynamics of very low concentrations of analytes and of singlemolecules. For many biologically relevant systems, however,micromolar concentrations of ligand are necessary for proper

enzyme function. FCS performed with far-field illuminationsuffers from small fluctuations at such large concentrations offluorescent ligand. Additionally, our group is currently pursuing a

strategy for single-molecule DNA sequencing that depends on

reducing the observation volume in order to follow the action ofDNA polymerase in real time. Using near-field apertures forillumination effectively reduces the observed volume and allows

for FCS at high concentrations. We have used arrays of 50 - 150nm apertures in aluminum films to perform FCS at highconcentrations. Experiments indicate that the effective volume ofobservation is well below one attoliter, making near-field aperturesa promising candidate for single molecule DNA sequencing.Finite-element calculations of the intensity distribution within theapertures were used to develop theoretical curves to fit to the data.

655.11-Pos Board # B535FLUORESCENCE DETECTION NEAR METAL LAYERSJoerg Enderlein; Regensburg University, PF 10 10 42,Regensburg, D-93051 GermanyImmobilization and self-assembly of biomolecules on noble metallayers is a widely used technique in bioanalytical and biomolecularstudies. Fluorescence detection is one of the most sensitivetechniques to detect and to study minute quantities of molecules.Unfortunately, due to strong electromagnetic quenching offluorescence emission by metals, it seems rather impossible tocombine the advantages of the immobilization techniques on noblemetal layers with the sensitivity and versatility of fluorescencedetection. Here, we present a thorough theoretical analysis offluorescence detection near metal layers on glass/quartz substrates,taking into full account all details of excitation and detection asmet in real experiments. It is shown that under carefully chosenconditions (evanescent excitation with specific incidence angle,excitation/emission wavelength, layer thickness, layer metal,molecule-surface distance), the presence of a metal has not to be ahindrance to sensitive fluorescence detection, but may be evenadvantageous. The presented quantitative results are important foranybody interested in sensitive fluorescence spectroscopy ofmolecules in the vicinity of metal layers.

655.12-Pos Board # B536SINGLE MOLECULE STUDIES OF LIVING COLOURS.M.F. Garcia-Parajo, M. Koopman, B. I. de Bakker, N.F. vanHulst; Applied Optics group, MESA Research Institute & Facultyof Applied Physics. University of Twente, P.O. Box 217, 7500AEEnschede, The Netherlands.The green fluorescent protein (GFP) has become an invaluablemarker for monitoring protein localisation and gene expression invivo. Mutagenesis of the original wild type GFP has lead tovariants fluorescing in colours ranging from blue to yellow. Theuse of these different mutants at the individual molecular levelrequires however a thorough study of their complexphotodynamical behaviour. Furthermore, in single pairfluorescence energy transfer (spFRET) experiments it is crucial todispose of pairs with emission spectra clearly separable and exactacknowledge of the emission dipole moments of bothchromophores. We have studied a number of GFP mutants at theindividual molecular level using a polarisation sensitive scanningnear-field optical microscope and have characterised their complexbehaviour. We present recent data concerning the photodynamicsof the S65T mutant, the RsGFP and the new red fluorescent proteindrFP583. A comparative study is presented in terms of theabsorption cross section, quantum efficiency, fluorescence lifetimeand blinking behaviour of the different mutants obtained fromsingle molecule data.

655.13-Pos Board # B537STUDY OF INDIVIDUAL TRANSMEMBRANE PROTEINSUSING A COMBINED CONFOCAL/NEAR FIELDOPTICAL MICROSCOPE.Baerbel I. de Bakker, M.F. Garcfa-Paraj6, W.H.J. Rensen, N.F.van Hulst, F. de Lange, A. Cambi, C.G. Figdor; Dept. of AppliedPhysics, University of TwenteCells are complex structures in which a wide range of processesoccur. By looking only at the cell membrane we already find alarge number of proteins with different functions: receptor proteins

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that allow cell-to-cell communication, transport proteins to carrymatter in-out of the cell, and marker proteins for cell identification.We focus particularly on the transmembrane receptor protein LFA-1. This protein plays an important role in the immune processregulating the adhesion strength of the cell via the formation ofclusters. We study both individual LPA-1 molecules as well astheir clustering. The LFA-1 molecule is fused to the GreenFluorescent Protein (S-65T mutant GFP) and expressed on L-cells.We have combined a confocal (CM) and a near field scanningoptical microscope (NSOM) to visualise individual LFA-1molecules at the cell membrane. We are also investigating thepacking density of the clusters, the distance between individualcomponents in a given cluster and cluster spacing distances. Oursetup allows a spatial resolution of 35 nm combined with singlemolecule detection sensitivity. In this contribution we discussinstrumentational issues regarding the CM/NSOM setup and showmost recent results.

655.14-Pos Board # B538BLINKING AND ITS EFFECT ON SINGLE MOLECULEFRET MEASUREMENTSHliroakl Yokota', Yoshiharu Ishii', Toshio Yanagida2; 'SingleMolecule Processes Project, ICORP JST, 24-14 Senba-Higashi,Mino, Osaka 562-0035 Japan, 2Single Molecule Processes Project,ICORP JST & Osaka University Medical School, JapanA repeated cycle of fluorescence turning-on and -off or blinkingbehavior has been observed for single nanoparticles and greenfluorescent protein (GFP) molecules. In this study, we found thatchemical fluorescent dyes such as CyS and IC5 also show blinking.In single molecule FRET (smFRET) measurements using thesefluorophores, the blinking effect should be taken intoconsideration, because the blinking of an acceptor results inapparent changes in the FRET efficiency. To distinguish theblinking from real FRET changes, the fluorescent state of anacceptor should be monitored at the same time. Donors(tetramethylrhodamine) and acceptors (IC5) were alternatelyexcited by switching laser light between at 532 nm and 633 nmusing electronic shutters. In addition, the donor and acceptorfluorescences were simultaneously imaged using a dual-viewapparatus and recorded by an ICCD video camera at a video rate of30 frames/sec. We found that some of smFRET efficiency changeswere due to the blinking of the accepter. Thus, we could eliminatethe blinking effect from smFRET measurements. After eliminatingthe blinking effect, we could analyze the FRET data from doublylabeled actin fixed on a glass surface.

655.15-Pos Board # B539SIMULTANEOUS "DUAL-COLOR AND DUAL-POLARIZATION IMAGING" AS A TOOL FOR CO-LOCALIZATION AND FLUORESCENCE RESONANTENERGY TRANSFER STUDIES OF SINGLE MOLECULESGerhard A. Blab, Laurent Cognet, Gregory S. Harms, Piet H. M.Lommerse, Thomas Schmidt; Leiden University, P.O. Box 9504,NL-2300 RA Leiden, NetherlandsVast progress has been achieved in the past decade in the study ofsystems by fluorescence on the level of single molecules. Standardprocedures allow one to follow a fluorescence labeled target with apositional accuracy of less than 50nm and on a timescale ofmilliseconds. Information about even closer proximity down to afew nanometer can be obtained by using fluorescence resonantenergy transfer (FRET). Specifically in life sciences, there is lessinterest in simply identifying biologically relevant substances in acell, rather the interest is in ascertaining the mobility, orientation

and the interaction of proteins in a membrane. Using a customizeddichroic wedge mirror, we were able to measure simultaneouslythe relative orientation, the emission and the position of twodifferent fluorophores (Cy5-streptavidin and TMR-biocytin) aswell as FRET by imaging at a rate of 10Hz. Applying thissimplified experimental routine to determine relative orientation ofcolocalized fluorophores, new insight into details of molecularinteraction as aggregation characteristics could be gained.

655.16-Pos Board # B540THE AGGREGATION OF INDIVIDUAL EYFP-LABELEDHUMAN CARDLIC L-TYPE CA" CHANNELS IN LIVINGCELL MEMBRANESGregory S. Harms', Gerhard A. Blabl, Piet H. M. Lommersel,Laurent Cognetl, Heike Kahr2, Roland Gamsjaeger2, NikolaiSoldatov3, Christoph Romanin2, Hermann P. Spainkl, ThomasSchmidtl; lLeiden University, P.O. Box 9504, NL-2300 RALeiden, NL-2300 RA Netherlands, 2Univ. of Linz, AltenbergerStr. 69, A-4040 Linz, Austria, 3NIH, 5600 Nathan Shock Dr.-Box29, Baltimore, MD 21224-6825L-type calcium channels are voltage-gated, multi-subunit proteinsinvolved in the regulation of calcium ion influx into muscle cells.To investigate the diffusion, confomational state andstoichiometry of these proteins in relation to their function, one hasto study these channels on the single molecule level. Here wepresent results obtained by two complementary techniques: wide-field imaging and fluorescence correlation spectroscopy. We firstdescribe the signal and mobility of the eYFP in cells and onmembranes. To observe the calcium channels, the a, subunit isfused to the enhanced Yellow Fluorescent Protein (eYFP). Usingwide field imaging, the diffusion of individual eYFP calciumchannel fusion proteins was observed in the membranes of livingcells. In addition, analysis of the local stoichiometry yieldedevidence for local aggregation of the channels. FluoresenceCorrelation Spectroscopy measurements were used to confirmthese results.

655.17-Pos Board # B541AN ARTIFICIAL LIPID BILAYER FORMED ONAGAROSE-COATED GLASS FOR SIMULTANEOUSELECTRICAL AND OPTICAL MEASUREMENT OFSINGLE ION-CHANNELS (II).Torm Idel, Toshio Yanagida2; 'JST, 2-4-14 Senba Higashi, Mino,Osaka 562-0035 Japan, 20saka UniversityThe purpose of this study is to develop an apparatus forsimultaneous measurement of electrical and spectroscopicparameters of single ion-channels. We combined the singlechannel recording apparatus with an artificial planar lipid bilayermembrane and a fluorescence microscope designed to detect singlefluorescent molecules. The artificial membranes were formed onan agarose-coated glass and observed with an objective-type totalinternal reflection fluorescence microscope. The membrane wasvoltage-clamped with a patch-clamp amplifier and single-channelcurrents were recorded simultaneously with the optical signals.Ionic channels were incorporated into the membrane and currentfluctuations were recorded at the single-channel level. Afterincorporation of Cy3-alamethicin molecules into the membrane,bright spots were observed moving rather slowly in the membrane,simultaneously with the alamethicin-channel current. Channelproteins from muscle cells were reconstituted into the membraneby vesicle-fusion and the electrical and optical properties weremeasured at the single channel level.

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655.18-655.22

655.18-Pos Board # B542SINGLE-CHANNEL PATCH-CLAMP RECORDINGCOUPLED WITH LINEAR AND NONLINEAR CONFOCALSCANNING FLUORESCENCE SPECTROSCOPY:TOWARDS THE SIMULTANEOUS PROBING OF SINGLE-ION CHANNEL CONFORMATIONAL CHANGES ANDCHANNEL KINETICS

Galya Orr1, Maurice Montal2, Brian Thrall', Steve Colson', H.Peter Lu'; 'Pacific Northwest National Laboratory, Richland, WA99352, 2Division of Biology, Section of Neurobiology, USCD, LaJolla, CA 92093

The measurements of ion-channel-protein conformational changescorrelated with ion-channel current trajectories are essential forunderstanding the mechanism and dynamics of ion channelfunction and structure in living cells. We have made significantprogress in developing a unique technology, by combining linearand non-linear fluorescence imaging spectroscopy with single-channel recording, patch-clamp technologies. The dimerizationdynamics of single-peptide ion channels, gramicidin A and dansylgramicidin C, were probed by monitoring single-ion channelcurrent on and off trajectories. The dynamics was found to beinhomogeneous due to different local environments around theindividual dimers and monomers. Extensive exploratoryexperiments have been carried out to obtain fluorescence single-ion channel imaging and to correlate dimerization dynamicsmeasurements from both fluorescence trajectories and ion currenttrajectories using the combined patch-clamp confocal microscope.Our approach is being applied to study the mechanism anddynanmics of single-glutamate receptors (AMPA and NMDAreceptors).Supported 'by the Laboratory Directed Research and DevelopmentProgram of Pacific Northwest National Laboratory operated for theU.S. Department of Energy (DOE) by Battelle and 2by NIH GM-49711 and USARO DAAG55-98-1-0106.

655.19-Pos Board # B543AUTOFLUORESCENT-PROTEINS IN SINGLE-MOLECULE RESEARCH: APPLICATIONS TO LIVECELL IMAGING MICROSCOPY

Gregory S. Harmsl, Laurent Cognet', Piet H. M. Lommerse',Gerhard A. Blab', Ewa Snaar-Jagalskal, Christoph Romanin2Nikolai Soldatov3, Herman P. Spaink', Thomas Schmidt'; 'LeidenUniversity, P.O. Box 9504, NL-2300 RA Leiden, NL-2300 RANetherlands, 2Univ. of Linz, Altenberger Str. 69, Linz, A-4040Austria, 3NIH, 5600 Nathan Shock Dr.-Box29, Baltimore, MD21224-6825

The spectral and photophysical characteristics of theautofluorescent proteins were analyzed and compared to flavinoidsin order to test their applicability for single-molecule microscopyin live cells. We compare: i) the number of photons emitted byindividual autofluorescent proteins in artificial and in vivosituations (about 3000 photons/ms), ii) the saturation intensities ofthe various autofluorescent proteins (from 6 to 50 kW/cm2), andiii) the maximal emitted photons from individual fluorophores inorder to specify their use for repetitive imaging and dynamicalanalysis, indicating photobleaching yields from 10-4 to I0-1.Definition of a detection-ratio led to the conclusion that theyellow-fluorescent protein mutant eYFP is superior among theautofluorescent proteins for single-molecule studies in vivo. Wehave applied single-molecule microsocpy using eYFP to a varietyof biological cell situations: 1) We have analyzed the self-aggregation of individual eYFP-labeled human cardiac L-type Ca2"channels in living cells. 2) We have observed single eYFP-CAAXdomain fusion proteins tethered to the plasma membrane to

investigate membrane inhomogeneities. 3) We have observedindividual eYFP-labeled -14-3-3 Map Kinases in living cellsinvolved in signaling pathways.

655.2-Pos Board # B544OBSERVATION OF A SINGLE DYE LABELED VIRUS ONITS INFECTIOUS ENTRY PATHWAY INTO A LIVINGCELLChristoph Brauchle; Ludwig-Maximilians-Universitat MtUnchen,Butenandtstr. 5-13, Haus E, MUlnchen, Bavaria D-81377 GermanyG. SEISENBERGER', T. ENDRESS', C. BRAUCHLE', M.RIED2, M. HALLEK2;'Institut fUr Physikalische Chemie andCenter for Nanoscience, Universitat Mtinchen, Butenandtstr. 11,81377 MtUnchen, Germany, 2 Genzentrum of the UniversititMtinchen, Feodor-Lynen-Str. 25, 81377 Mtinchen, GermanySingle molecule experiments were conducted to follow themigration of single adeno-associated viruses on their infectiousentry pathway into living HeLa cells. Only one fluorescent dyemolecule (Cy5) was attached to the viruses in order not toinfluence their physiological behavior. For the first time we couldobtain diffusion trajectories of single viruses in four differentstages of the infection:1.) Diffusion of the virus in front of the cell membrane andreceptor mediated adsorption2.) Formation of the endosome with inclusion of the virus3.) Transport of the endosome in the cell plasma and release of thevirus

4.) Penetration and diffusion of the virus in the cell nucleusFrom the trajectories of the individual viruses diffusioncoefficients were obtained for all four stages. A detailed picture ofthe processes involved was modeled. Adeno-associated virusesshow promising prospects for the use in human gene therapy. Forthis purpose a detailed understanding of the interactions of thevirus and the target cell is important.

655.21-Pos Board # B545UNRAVELING THE ELECTRONIC STRUCTURE OFINDIVIDUAL PHOTOSYNTHETIC PIGMENT-PROTEINCOMPLEXESAntoine M. van Ojen, Martijn Ketelaars, Michio Matsushita,JtUrgen Kohler, Thijs J. Aartsma, Jan Schmidt; Leiden University,P.O. Box 9504, Leiden, 2300 RALow-temperature single-molecule spectroscopic techniques areapplied to a light-harvesting pigment-protein complex (LH2) frompurple photosynthetic bacteria. The properties of the electronicallyexcited states of the two circular assemblies (the 9-memberedB800 and 18-membered B850 rings) of bacteriochlorophyll (BChla) pigment molecules in the individual complexes are revealed,without ensemble averaging. The results show that the excitedstates of the B800 ring of pigments are mainly localized onindividual BChl a molecules.' In contrast, the absorption of aphoton by the B850 ring can be consistently described in terms ofan excitation that is completely delocalized over the ring.2 Thisproperty may contribute to the high efficiency of energy transfer inthese photosynthetic complexes. Moreover the spectra indicate thatthe B850 assembly has an elliptical rather than a ring-likestructure. The results demonstrate the possibility of single-molecule spectroscopy to reveal in detail the factors determiningthe electronic structure of photosynthetic light-harvesting systemsand, more generally, of pigment-protein complexes.('van Oijen et al., Biophys.J. 78 (2000) 1570; 2 van Oijen et al.,Science 285 (1999) 400)

655.22-Pos Board # B546PHOTON ARRIVAL TIME INTERVAL DISTRIBUTIONANALYSIS (PAID) A NEW METHOD TOSIMULTANEOUSLY ANALYZE BRIGHTNESS,DIFFUSION TIME, AND CONCENTRATION OFBIOMOLECULES IN SOLUTIONTed Alfred Laurence', Daniel S. Chemla', Shimon Weiss2; 'UCBerkeley, LBNL, MS 2-300, 1 Cyclotron Road, Berkeley,

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California 94720, 2Lawrence Berkeley National Lab, MS 2-300, 1Cyclotron Road, Berkeley, California 94720

PAID is a new method for simultaneous determination ofbrightness, diffusion time, and concentration of fluorophore-labeled molecules undergoing diffusion in a confocal detectionvolume. This method is based on recording the time of arrival ofall detected photons, and then plotting the two-dimensional

histogram of photon pairs, where one axis is the time between twophotons and the second axis is the number of other photonsdetected within that photon pair interval. It is related toFluorescence Correlation Spectroscopy (FCS) by a projection ofthis histogram onto the time axis. PAID extends FCS bymeasuring brightness in addition to diffusion time. Other methods,such as Fluorescence Intensity Distribution Analysis (FIDA) andPhoton Counting Histogram analysis (PCH), measure brightness,but not diffusion time. We are developing data-fitting analysisbased on Monte Carlo simulations of diffusion paths through thedetection volume which we will use to extract brightness, diffusiontime, and concentration from experimental and simulated data. Weare testing the ability of PAID to separate sub-populations withdifferent brightness and/or diffusion times in solution. We will

compare the performance of this method with FCS for extractingdiffusion time and with FIDA and PCH for extracting brightness.

655.23-Pos Board # B547EXCITON STATES OF AN ELLIPTICALLY DEFORMEDL1I2 RING.ThiJs J. Aartsmal, Micho Matsushita2, Martijn Ketelaars', MichoMatushital, Antoine M. van Oijen2, Juergen Koehler3, JanSchmidt2; 'Leiden University, P.O. Box 9504, Leiden, 2300 RANetherlands, 2Leiden University, Department of MolecularPhysics, 3University of Bayreuth, Bayreuth, GermanyFrom the analysis of the single molecule spectra of the LH2complex of Rhodopseudomonas acidophila 1.2 K it wasconcluded that, in addition to the random disorder, a regularmodulation of the interaction had to be assumed to explain theobserved spectra. This modulation has a C2 symmetry whichstrongly suggests a structural deformation of the ring into an

ellipse. Here we will discuss and compare three different modelsfor an elliptical deformation of the B850 ring, aimed at providinga link between the geometrical deformation and the spectroscopicproperties of the individual LH2 complex. With the simple point-dipole approximation, an elliptical ring of b/a = 0.85 can

reproduce the splitting of the k = ±1 exciton states, while theirmutual orthogonahty is maintained. However, the intensity ratiobetween the two k = +1 components depends on the arrangementof the pigments on the ellipse, and is compatible with a model inwhich the inter-pigment distance is shortest at the short axis of theellipse. The pigments are closer to one another where thecurvature of the ellipse is small. This model is also consistent withthe weak polarization dependence of the k = +3 band. Thus, withone free parameter for the ellipticity we can explain the majorfeatures of the spectra of individual LH2 complexes.

655.24-Pos Board # B548SINGLE-MOLECULE CONFORMATIONAL DYNAMICSOF PROTEINS.

Pallop Karnchanaphanurach', Haw Yang', Tai Man Louie2,Luying Xun2, X. Sunney Xie'; 'Harvard University, 12 OxfordStreet, Cambridge, MA 02138, 2Washington State University,Pullman, WA 99164-4234The dynamical changes of protein conformation can be either fastor slow, spanning from femtoseconds to seconds. While the fastfluctuation can be probed by ultrafast spectroscopic techniques, theslow fluctuation is commonly hidden in ensemble averagedmeasurements. We have developed a new approach to studyconformational dynamics on microsecond to second time scales.

The fluorescence lifetime of FMN provides a distance-dependentprobe for conformational changes. Measurements on singleNADH:flavin oxidoreductase (Fre) molecule from E. coli indicatea broad distribution of conformational states and dynamicalfluctuation among them. Enzymatic turnovers of single Fremolecules are also observed in real time, offering an opportunity tostudy the influences of conformational fluctuation on the reactiondynamics.

655.25-Pos Board # B549SEQUENCE-DEPENDENT PAUSES OBSERVED FORSINGLE LAMBDA EXONUCLEASE MOLECULESThomas T. Perldns', Paul G. Mitsis2, Ravindra V. Dalal', StevenM. Block'; 'Stanford Univ., Stanford, CA 94305, 2Praelux,Lawrenceville, NJ 08646Lambda exonuclease (A-exo) is a donut-shaped homotrimer thatprocessively degrades one strand of double-stranded DNA in the5-3' direction. We developed a single molecule assay for X-exoanalogous to the existing tethered particle geometry for RNApolymerase. X-exo was attached to a coverslip while bound to oneend of a DNA molecule which had a 0.46 gm dia. bead coupled tothe distal end. Motion of X-exo along the DNA template wasdetermined using back focal-plane detection of scattered light froman optically-trapped bead. A force clamp was implemented bymoving a 3-axis piezo stage programmatically in response toenzymatic motion. High precision measurements of enzymeposition (-10 nm) were achieved by minimizing long-terminstrumental drift (<10 nm over 600 sec) and noise (<0.1 nm/4Hzfor v > 0.3 Hz). X-exo location along the DNA template wascomputed by determiining the enzyme tether position to <10 nmwith a protocol that found the symmetry point in the DNAelasticity measured on either side of its attachment site. Verticalposition was determined to -5 nm by measuring changes inscattered light that occur when the bead makes contact with thecoverslip. Variations in trap stiffness with depth were -2 % over.500 nm, and variations in lateral sensitivity were reduced to <2%for -100 nm vertical motion within the trap. These instrumentalimprovements allowed us to use the assay to resolve sequence-dependent pauses in the motion of X-exo. The origin andnucleotide sequence of these pauses is being investigated.

655.26-Pos Board # B550EXPLORING THE ION-FACILITATEDCONFORMATIONAL CHANGE OF A SINGLE RNAMOLECULEHarold D. Kim', G. Ulrich Nienhaus2, Taekjip Ha3, Jeffrey W.Orr4, James R. Williamson4, Steven Chu'; 'Stanford University,2University of Ulm, 3University of Illinois, 4The Scripps ResearchInstituteFluorescence correlation spectroscopy on immobilized singlefluorophores was developed to study the Na+ or Mg2e-facilitatedconformational change of an RNA triple-helix junction thatinitiates the folding of the 30S ribosomal subunit. Transitionsbetween open and folded conformations were monitored byfluorescence resonance energy transfer (FRET). Fluorescencecorrelation analysis was applied to fluctuating fluorescence signalsfrom single RNA molecules in order to obtain folding and openingrates of RNA junctions over a wide range of concentrations. Wefound that Nae and Mg2e both facilitate folding with equilibriumdissociation constants of 350mM and 140gM, respectively. It wasalso found that the opening rate varies much more dramaticallythan the folding rate as cation concentration is varied. These resultssuggest that the conformational change might be due to cationstabilization of RNA junction through electrostatic screening.

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655.27-655.31

655.27-Pos Board # B551STRUCTURE AND DYNAMICS OF CLUSTERS OF C5ARECEPTORS FORMED IN RESPONSE TO APPLICATIONOF C5A TO LIVING HEK-293 CELLSStuart Licht', Alois Sonnleitner2, Daniel S. Chemla2, Peter G.Schultz', Shimon Weiss2; 'The Scripps Research Institute, La Jolla,CA 92037, 2Lawrence Berkeley National Laboratory, Berkeley,CA 94720In order to study dynamic redistribution of G protein-coupledreceptors associated with receptor activation and clathrin-mediatedendocytosis, total internal reflection microscopy was used to obtainimages of living HEK-293 cells co-expressing a C5a receptor-yellow fluorescent protein (C5aR-EYFP) fusion and a clathrin-redfluorescent protein (RFP) fusion. In the absence of agonist, C5aR-EYFP was uniformly distributed in the plasma membrane, butwhen the agonist C5a was added at a saturating concentration,C5aR-EYFP was observed to form large assemblies co-localizingwith clusters of clathrin-RFP, as judged by dual color imaging.The assemblies varied in size, with the largest producing imagesizes of >1 um2. Imaging with 100 ms frame rate showed that theassemblies often exhibited dramatic fluctuations in fluorescenceintensity on the timescale of seconds, consistent with a change in

the number of receptors in the cluster and/or motion of thereceptors away from the plasma membrane due to invagination ofcoated pits or vesicle formation. The structure and dynamics ofthese clusters of receptors may provide new insights into themechanism of clathrin-mediated endocytosis.

655.28-Pos Board # B552MOBILITY OF LIPID-ANCHORED PROTEINS IN A CELLMEMBRANE VISUALIZED BY SINGLE-MOLECULEMICROSCOPYPiet H.M. Lommerse, Laurent Cognet, Gerhard A. Blab, Greg S.Harms, Ewa Snaar-Jagalska, Herman P. Spaink, Thomas Schmidt;Leiden University, Niels Bohrweg 2, Leiden, 2333 CANetherlandsA variety of proteins are attached to the cell membrane via lipidanchors. The mobility and organization of these proteins in thecell membrane is thought to play a key role in signal transductionprocesses. Some studies indicate the possibility that these proteinsare preferentially located in subdomains of the cell membrane thathave a high cholesterol and glycolipid content, called 'rafts'. Inthis study, the lateral diffusion of lipid-anchored proteins wasstudied in the membrane of live cells using wide-field single-molecule microscopy. As a model for lipid-anchored proteins, thehuman H-Ras protein was selected. This protein is linked to thecell membrane via farnesylation and palmitoylation of cysteineresidues at the C-terminus (CAAX domain). To be able to observethe lateral diffusion of these lipid anchors, the C-terminus of H-Ras was fused to the C-terminus of the enhanced YellowFluorescent Protein (eYFP), resulting in plasma membranelocalization of eYFP. Using single-molecule microscopy the lateralmobility of anchored eYFP was studied in the cell membrane oflive tsA201 cells. To investigate the influence of specific protein-protein interactions on the mobility, results are compared to themobility of eYFP-H-Ras fusion proteins.

655.29-Pos Board # B553ANOMALOUS DIFFUSION IN DENDRITES OFCULTURED NEURONS REVEALED BY FLUORESCENCECORRELATION SPECTROSCOPY.Arne Gennerich, Detlev Schild; Institute of Physiology,Humboldtallee 23, Gottingen, 37073 GermanyFluorescence correlation spectroscopy (FCS) can be used tomeasure kinetic properties of single molecules in drops of solution

or cells. Here we report on FCS measurements of TMR-dextran(10 kDa) in dendrites of cultured neurons. To interpret suchmeasurements appropriately, the plasma membrane boundaries ofthe dendrites have to be taken into account. We show that thefluorescence data recorded from dendrites are best described by amodel of confined anomalous diffusion. Our analysis suggest thatdiffusion in neural dendrites is anisotropic in the sense that it isnormal along the dendrite and anomalous in the directionorthogonal to the dendritic extension. Along the dendrite,molecules appear to diffuse about one order of magnitude fasterthan across the dendrite.

655.3-Pos Board # B554A-RECEPTOR DIFFUSION STUDIED WITH OPTICALTWEEZERSKirstine Berg-Sorensen', Lene B Oddershede2, Sonia Grego3,Jakob K Dreyee2, Stanley Brown4; 'Nordita, Blegdamsvej 17,Copenhagen, 2100, 2Niels Bohr Institute, 3University of SC atChapel Hill, 4Dept. of Mol. Biology, Copenhagen UniversityWe study the mobility of the X-receptor in the membrane of E. Colibacteria. The k-receptor has been genetically modified to allow forbiotinylation at an extra-cellular site. Streptavidin coated gm sizedpolystyrene beads are then coupled to the k-receptors. As theefficiency of the biotinylation is low, it is likely that there is nomore than one X-receptor per bead. We intend to measure theviscosity of the bacterial membrane felt by the ?-receptor in a localmeasurement: The polystyrene bead is held in a soft opticalpotential. Its stochastic motion is determined by a combination ofthe diffusion of the protein, diffusion of the bead itself, and theaction of the optical potential. We analyse this motion to obtaininformation on the diffusional properties of the k-receptor. Part ofour analysis is also relevant for single particle trackingexperiments.

655.31-Pos Board # B555DIRECT MEASUREMENTS OF MULTIPLE ADHESIVEALIGNMENTS BETWEEN CADHERINEXTRACELLULAR DOMAINS.Deborah E. Leckband', Sanjeevi Sivasankar', Barry Gumbinee,Sophie Chapuis-Flament2, Laurence Martel3, Jean-FrancoisLegrand3; 'University of Illinois, 600 S. Mathews Ave., Urbana, IL61801, 2Memorial Sloan Kettering Cancer Center, 1275 YorkAve., New York, NY 10021, 3CNRS-Grenoble, 17 rue desMartyrs, 38054 Grenoble, Cedex 9 FranceCadherins are large multidomain proteins that mediate cell-celladhesion in all soft tissue. The extracellular domains undergohomophilic adhesion with identical proteins on the opposingsurface, but the trans interactions responsible for adhesion betweencadherins have not been identified. Here we report theidentification of three antiparallel adhesive protein alignments thatmediate binding. In addition to the force measurements, whichidentified the binding configurations, we describe the results of X-ray reflectivity measurements, which confirm the orientation of theextracellular domains on supported bilayers. Finally, forcemeasurements using domain deletion mutants were done toidentify the force-generating protein domains responsible for eachof the three antiparallel binding orientations.

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65532-Pos Board # B556UNBINDING OF SINGLE PHOSPHOLIPASE A2MOLECULES FROM LIPID BILAYERSLars Kildemark Nielsen', Hauke Clausen-Schaumann2, MichelGrandbois2, Herman Gaub2, Ole G Mouritsen1; 'TechnicalUniversity of Denmark, Building 207, Lyngby, DK-2800Denmark, 2Univeristy of Munich, Amalienstr. 54, Munich, D-80799 GermanyIn order to perform any hydrolysis of phospholipids phospholipaseA2 must bind to a lipid membrane. We have investigated thebinding strength of single PLA2 molecules using dynamic forcespectroscopy. By functionalizing an AFM cantilever with PLA2 theunbinding force of individual PLA2 molecules from phospholipidbilayers can be measured. The results show that unbinding ofsingle molecules can be detected for DMPC bilayers. Themeasured force displays a logarithmic dependence on the force-loading rate indicating that the unbonding is dominated by a singlepotential barrier. The interaction also shows a dependence on thelipid head-group charge leading to increased unbinding force forthe negatively charged lipid DSPG.

655.33-Pos Board # B557SINGLE-MOLECULE FORCES BETWEEN c4upINTEGRINS AND GRGDSP PEPIIDE-AMPHIPHILESEfrosini Kokkoli, Matthew Tirrell; University of California atSanta Barbara, Santa Barbara, CA 93106We report on single-molecule force experiments that reveal detailsof the molecular recognition mechanisms of individual ligand-receptor pairs. This study involves the use of a model biomimeticsystem that allows us to investigate unbinding processes between asingle o4fil-GRGDSP pair. An Atomic Force Microscope (APM)is used to provide high resolution images and adhesionmeasurements at the piconewton level. In this work, bioartificialmembranes that mimic the tenth type m module of theextracellular matrix protein fibronectin (GRGDSP) are constructedfrom peptide-amphiphiles. GRGDSP is the primary recognitionsite for Nos,l and peptide-amphiphiles containing the GRGDSPsequence are deposited on a surface by the Langmuir-Blodgetttechnique. The receptor of choice is the aspi integrin. Twodifferent antibodies have been used to activate and immobilizeisolated acfci integrins on the AFM tip. The effect of differentparameters such as synergistic effects from other peptides, theeffect of different ions, the loading rate have been investigated onthe dynamics of the Nctsio-GRGDSP interaction using the AFM.

655.34-Pos Board # B558THE EFFECT OF EXTERNAL LOADS ON SINGLEMYOSIN-ACTIN CROSSBRIDGESAndrea L. Stout; Swarthmore College, 500 College Ave.,Swarthmore, PA 19081The advent of single-molecule techniques has prompted muchinterest in the effects of mechanical loads and loading rates on thedissociation kinetics of intermolecular interactions. One interactionthat may show load-dependent behavior is that between MgADP-smooth muscle myosin heads (SI) and actin. Using an opticaltweezers equipped with sensitive position/force measurement andfluorescence imaging capabilities, experiments are underway tolook at the effect of applied loads on the lifetime of actin-(MgADP-smSl) bonds in the presence of varying ADPconcentrations. Monitoring the displacement signals from anoptically-trapped smSl-coated bead while a surface-immobilizedactin filament is scanned beneath it allows one to detect theformation and breakage of individual bonds. The effects of bothloading rate and loading direction on the mean breakage force andaverage bond lifetime can then be quantified. (Supported byCottrell College Science Award No. CC4911 from ResearchCorporation.)

655.35-Pos Board # B559TWO-STATE MODEL OF POLYSACCHARIIDEELASTICITY PROBED WITH THE FORCE CLAMPMODE OF AFMPlotr E Marszalek, Andres F Oberhauser, Julio M Fernandez,;Mayo Foundation, 1-117 Medical Sci. Bldg., Rochester, Minnesota55905We recently showed that polysaccharide elasticity is governed byforce-induced conformational transitions within the pyranose ring(e.g. chair-to-boat transitions). Typically, we measure the elasticity

500 of polysaccharides by4w stretching their single

I 7w molecules in the atomic forceI 200 11 _ i microscope (AFM). TheseJ 1ao g experiments produce force

extension curves which0 ~ display a prominent plateau80 0 80 1000 1800 0 which is not accounted for by

FamW* simple models of entropicelasticity (freely jointed chain). Here we derive an analyticalsolution for the extensibility of a polysaccharide chain based on asimple two-state model of its segments (short segment-chairconformation; long segment-boat conformation) with the rateconstants of the chair-to-boat and the boat-to-chair transitionreaction explicitly depending on the applied force. To test this two-state model we developed a new mode of the operation of the AFMthat, through a feedback system, is capable of extending thepolymer with a linearly increasing force (force ramp). We find thatthe model accurately reproduces the plateau feature in the force-extension relationship of polysaccharides and allows us todetermine their physico-chemical characteristics, such as segmentelasticity and rate constants of the conformational transitions.Supported by the NSF and the NIH.

655.36-Pos Board # B560STEPWISE UNFOLDING OF TITIN UNDER FORCE-CLAMP AFMAndres F Oberhauer1, Paul K Hansma2, Mariano Carrion-Vazquez', Julio M Fernandez', ; 'Mayo Foundation, Rochester,MN 55905, 2University of California Santa Barbara, CAThe mechanical extension of the muscle protein titin is thought tobe due to probabilistic unfolding events that are dependent on thestretching force. Here we demonstrate the implementation of asingle molecule force-clamp adapted for use with an AFM that weuse to directly test this hypothesis. Force-clamp recordings give

350 exceptionallyclean records

300 ~~~Of the250 2WpN 9 °fmechanical

't 200 O pN sH-unfolding of150 ~~~~~~~~~titin. We show

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sou J conditions, an0 - engineered titin

0 1 2 3 4 5 6 7 proteinfime (s) elongates in

steps due to the unfolding of its modules (see figure) and that thewaiting times to unfold are exponentially distributed, as predictedby a simple first order kinetic model. We also show that theunfolding probability depends sigmoidally on the applied force.Single molecule force clamp measurements, as demonstrated herepermit, for the first time, direct measurements of the force-drivenunfolding kinetics of a single protein. This new fonn of singleprotein force spectroscopy promises to be a direct way to probeelastic proteins such as those found in muscle, the extracellularmatrix and in cell adhesion.

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655.37-Pos Board # B561SINGLE MOLECULE STUDY OF HELICASECONFORMATIONAL CHANGES AND DNA UNWINDINGTaekjip Hal, Hazen P Babcock2, Wei Cheng3, Timothy MLohman3, Steven Chu2; 'University of Illinois, 1110 W. Green,Urbana, IL 61801, 2Stanford University, 382 via Pueblo Mall,Stanford, CA 94305 United Arab Emirates, 3WashingtonUniversity School of Medicine, St. Lous, MO 63110 United ArabEmiratesNucleic acid unwinding, catalyzed by helicases, is essential formany cellular processes. Conformational changes of helicaseinduced by nucleotide binding, hydrolysis and release propel itsmotion along nucleic acids and its unwinding action, hencehelicases are considered molecular motors. Fluorescence resonanceenergy transfer between two fluorophores attached to DNAsubstrate was used to detect the changes of individual DNAmolecules induced by helicase binding, conformational change andunwinding action. We detected two distinct conformational statesof E.coli. helicase Rep monomer bound to a DNA that were notexistent for another E.coli. helicase UvrD. Conformationaldistribution and fluctuation rates were strongly dependent onnucleotide bound states, indicating they reflect the conformationsof the helicase and are not of purely DNA origin. In particular,conformational fluctuation rate increases at least ten fold in thepresence of ATP, implying the functional roles of the fluctuations.We also detected unwinding intermediates (only a few base pairsunwound) induced by Rep helicase and hence clearly distinguisheda partial or transient unwinding from a complete unwinding.Inplications of this work in relation to recent structural andbiochemical studies on helicases are also discussed.

655.38-Pos Board # B562MASSIVELY PARALLEL MEASUREMENTS OFMOLECULE/SURFACE BINDING FORCESFabiano Assi, Rob Jenks, Gary Zabow, Mara Prentiss; HarvardUniversity, 146 Jefferson Lab, Cambridge, MA 02318There is great interest in examining the binding process that attachsingle molecules to surfaces. Various tools have been used toinvestigate these interactions, including optical tweezers, magnetictweezers, and atomic force microscopes; however, most of thetools have allowed only one measurement at a time. We reportexperiments that can make thousands of measurements in parallelat one time. These experiments use superparamagnetic beadswhose surfaces have been treated to accept binding by moleculessuch as DNA. Carefully tailored magnetic fields can then controlthe position and the force exerted simultaneously on thousands ofbeads, where the magnetic fields can be produced either bypermanent magnetic materials or by current carrying wires. In onesimple configuration, arrays of alternating magnets can exertforces larger than a nano-Newton uniformly over a region with anarea of several square centimeters. An alternate configurationexploits the magnetic dipole/dipole interaction between theparamagnetic beads to self-assemble an ordered crystal lattice ofbeads at an air water interface. The lattice spacing in the crystalcan then be controlled, allowing measurements of adhesionprobability and strength as a function of separation distance, time,and temperature.

655.39-Pos Board # B563RIGIDITY OF TYPE I PROCOLLAGEN MOLECULEYulong Sun, Zong-Ping Luo, Kai-Nan An; Mayo Clinic, 128Guggenheim Building, Rochester, MN 55905Collagen is the major component of extracellular matrix providingmechanical and supportive functions. The significance of theknowledge in the mechanical behavior of the collagen moleculeshas been recognized in order to understand the etiology of tissuedegeneration and the mechanism of regeneration. This studydirectly measured the rigidity of type I collagen molecules. The

biomechanical tests of type I collagen were performed using an

optical tweezer/interferometer device. Type I procollagen was usedto attach polystyrene beads to its extension peptides, one at eachend, for the mechanical tests. The force-deformation relationshipof procollagen molecules was fitted into a worm-like chainelasticity model. The molecule length and persistence length oftype I procollagen were 293.1+33.6 and 58.0±8.7 nm (n=15),respectively. This investigation, to our knowledge, is the firstdirect quantification of the mechanical characteristics of collagenat the molecular level. Such a technique has great potential inextracellular matrix biomechanics.

655.4-Pos Board # B564A COMBINED ATOMIC FORCE, CONFOCAL, ANDTOTAL INTERNAL REFLECTION MICROSCOPE FORTHE STUDY OF SINGLE MOLECULESWilliam Frederick Heinz, Bertrand Navarro, Kenneth D. Weston,Pernelle Bernardi, Lori S. Goldner; NIST, 100 Bureau Drive, MailStop 8442, Gaithersburg, MD 20899-8442The interactions between biological macromolecules andmacromolecular assemblies are central to the proper functioning ofbiological systems. Historically, these interactions have beencharacterized by techniques that measure the averaged interactionsof large ensembles of particles. Recently, a number ofexperimental approaches have been developed that enable thestudy of single molecules, thus the distributions that make up theaverage behavior and the heterogeneity of a population can bedetermined as well as the time-dependent behavior of themolecules. Interactions between individual molecules depend onmany variables including orientation, conformation, localenvironment, and separation distance. We propose to monitormolecular configuration or separation as a function of force on themolecule by combining single molecule force measurements withsingle molecule optical techniques. Here we present an instrumentthat combines AFM or optical tweezers with confocal and totalinternal reflection microscopy and report on progress towards thesimultaneous measurement of force and orientational andconformational dynamics of single bio-molecules in aqueousconditions.

655Al-Pos Board # B565RNA TERTIARY AND SECONDARY UNFOLDINGMONITORED BY MECHANICAL STRETCHING OFSINGLE MOLECULESGloria Bibiana Onoa, Jan T. Liphardt, Steven B. Smith, IgnacioTinoco, Carlos J. Bustamante; University of California, Berkeley,419 Latimer Hall, Berkeley, CA 94720The pathways by which large RNAs adopt tertiary structure are ofcurrent interest, so new methods that reveal steps in RNAfolding/unfolding are highly desirable. We mechanically unfoldindividual Tetrahymena thermophila ribozyme molecules usingforce-measuring optical tweezers. Force-extension curves forunfolding of the 160-nucleotides P4-P6 domain reveal severaldistinct Mg2-dependent kinetic barriers. These barriers arecharacterized from non-equilibrium unfolding experiments, andseveral barriers are mapped to ribozyme domains usingcomplementary oligonucleotides targeted to candidate sequences.Unfolding of the L21 group I intron occurs along several differentpaths as revealed by differing force-extension curves showing thekinetic barriers, which represent the unfolding of differentsecondary and tertiary elements. At least 14 intermediate kineticbarriers are seen, which can be grouped into three clustersaccording to their re-folding kinetics and their positions on theforce-extension curves. Comparison of the signals from theisolated P4-P6 domain with the entire ribozyme indicates that P4-P6 is the last region of L21 to unfold. The most prominent signalof L21's unfolding curves identifies the unfolding of the P5abcsubdomain.

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655.42-Pos Board # B566FORCED UNFOLDING MODULATED BY DISULFIDEBONDS IN AN IMMUNOGLOBULIN CELL ADHESIONMOLECULEPhilippe Carl, Gavin Manderson, David Speicher, DennisDischer; University of Pennsylvania and Structural Biology,Wistar InstituteDisulfide modulation of protein unfolding under force has beendemonstrated with an Immunoglobulin (Ig) cell adhesion molecule(CAM). The particular protein studied, MeICAM, is highlyexpressed in melanoma cells; it is otherwise typical of the manyCAM family members in having tandem beta-barrel Ig domainsthat are all oxidatively stabilized by intradomain disulfide bonds.Mechanical unfolding of dithiothreitol-reduced, recombinantMelCAM domains has been accomplished through an impulsivemethod of atomic force microscopy analogous to that originallyapplied to the cytoskeletal protein titin (Rief et al, 1997). Underreducing conditions, we find a force for full unfolding of 30 - 50pN. This is considerably less than the 100 - 200 pN forcesreported either for Ig domains of titin or similar type-HIfibronectin domains, neither of which are stabilized by disulfidebonds. Moreover, under oxidative conditions, partial unfolding ofMeICAM domains is suggested by even smaller forces and shorterunfolding lengths. This is consistent with primary sequence whichshows that disulfide-linked cysteines bracket and stabilize abouthalf of each Ig domain rather than the entirety of each domain.The results point to the important contributions that disulfidebridges can make to the overall compliance of proteins, includingadhesion molecules that are invariably stressed in cell attachment.

655.43-Pos Board # B567FINGERPRINTING POLYSACCHARIDES WITH SINGLEMOLECULE ATOMIC FORCE MICROSCOPYPiotr E Marszalek, Hongbin Li, Julio M Fernandez, ; MayoFoundation, 1-117 Medical Sci. Bldg., Rochester, Minnesota55905We report the use of a novel AFM-based force spectroscopytechnique to identify, at the single molecule level, the composition

2500

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of mixtures of polysaccharides. Previously, we showed that theelasticity of certain types of polysaccharides is governed by force-induced conformational transitions of the pyranose ring. Thesetransitions produce atomic fingerprints in the force-extensionspectrum that are characteristic of the ground energy conformationof the pyranose ring and the type of glycosidic linkages. By usingthis approach we find that commercially available agarose and Xcarrageenan contain molecules that, when stretched in an atomicforce microscope, produce a force spectrum characteristic of a-(1,4) D-glucans. We have identified these molecules asamylopectin or Floridean starch, a storage sugar in algae. Ournovel methodology can be used to identify individualpolysaccharide molecules in solution which is not possible by any

other spectroscopic techniques. Supported by the NSF and theNIH.

655.44-Pos Board # B568MECHANICAL UNFOLDING OF INDIVIDUAL T4LYSOZYME MOLECULESGuoliang Yang', Ciro Cecconi2, Walter A Baase3, Brian W.Matthews3, Rick Dahlquist3, Carlos Bustamante2; 'DrexelUniversity, Philadelphia, PA 19104, 2University of California atBerkeley, Berkeley, CA 94720, 3University of Oregon, Eugene,OR 97403Single molecule manipulation techniques have offered a newapproach to investigate the protein folding problem. So far, mostof these studies are done on naturally-occurning polymeric proteinswith mechanical functions. To extend these techniques to studyglobular proteins in general, a method was developed to synthesizepolymers of protein molecules in the solid state. By inducingdisulfide bond formation between adjacent molecules in thecrystal, polymers of bacteriophage T4 lysozyme molecules up to25 molecules long were obtained. The polymers, which retain theenzymatic activity and have a defined polarity, were thenmanipulated mechanically using a modified scanning forcemicroscope to characterize the mechanical unfolding of individuallysozyme molecules. The force required to unfold the monomerswas measured and the refolding upon relaxation was characterized.The data suggest that the force-induced unfolding transition of T4lysozyme follows a two-state model. To help with theinterpretation of experimental data and to serve as a guide foroptimization of the experimental parameters, Monte Carlosimulations of the mechanical unfolding process were alsoperformed. The simulation results show that certain precautionshave to be taken when analyzing the experimental data andselecting a set of appropriate experimental parameters.

655.45-Pos Board # B569ENHANCED COOPERATIVITY AMONG LIGAND-RECEPTOR BONDS RESULTS FROM INCREASES INRETRACTION RATE DURING FORCE-INDUCEDDISSOCIATION.Xiaohul Zhang, Vincent T. Moy; University of Miami, 1600NW1Oth Avenue, Miami, FL 33136Cooperativity of molecular adhesion has been proposed as amechanism for enhanced binding strength between adhesionmolecules on apposing cell surfaces. We have previously shownthat the cross-linking of cell surface receptors can lead to greatercooperative binding. However, it has not yet been determined howthe unbinding rate of force-induced ligand-receptor dissociationaffects binding cooperativity. We addressed this question by directforce measurements using Atomic Force Microscopy (AFM). Theforce measurements were carried out using an avidinfunctionalized cantilever tip and a biotinylated agarose bead underconditions where multiple avidin-biotin linkages were formedfollowing surface contact. At slow retraction (200-S00nm/s) of theAFM cantilever from the bead's surface, the avidin-biotin linkagesappeared to rupture sequentially. Increasing the retraction ratefrom 200 to 2000nm/s led to an approximately linear enhancementin the average rupture force. Moreover, force histograms revealeda quantized force distribution. Increases in the retraction rate led toa shift in the force distribution from lower to higher values.Together, these results demonstrate that the cooperativity ofligand-receptor bonds is significantly enhanced by increasing theretraction rate.

655.46-Pos Board # B570PROBING THE INTERACTION BETWEEN LEUKOCYTEFUNCTION-ASSOCIATED ANTIGEN-1 (LFA-1) AND

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INTERCELLULAR ADHESION MOLECULE-1 (ICAM-1)BY CELL-CELL DE-ADHESION FORCESPECTROSCOPY.Xiaohui Zhang, Ewa Wojcikiewicz, Vincent T. Moy; Universityof Miami, 1600 NW10th Avenue, Miami, FL 33136Cell-cell adhesion plays a crucial role in many cellular processes,including regulation of the immune response of lymphocytes toantigen-bearing cells. Here we measured cell-cell de-adhesionforce by coupling a T cell hybridoma (3A9) to an Atomic ForceMicroscopy (AFM) cantilever, then measuring the force associatedwith its interaction with FTl6.I1 cell, a fibroblast cell linetransfected with the gene for ICAM-1. At the start of the forcemeasurement, the 3A9 cell was lowered on a FP16.11 cell toinitiate cell-cell contact and adhesion. The T cell was subsequentlywithdrawn from the FT16.11 cell while the force spectrum of theprocess was recorded. A distribution of rupture forces in the rangeof 30 to 150 pN was detected from force histograms. Comparisonof measurements carried out with ICAM-1 positive and negative(Frl6.6C5) cells further revealed a peak centered at approximately80 pN that can be attributed to the interaction between LFA-1 andICAM-1. The addition of antibodies against LFA-1 or ICAM-1reduced this peak in force distribution. Moreover, T cell treatedwith phorbol ester exhibited enhanced adhesion over the entirerange of the force histogram. In contrast, EDTA or EGTAsignificantly reduced cell-cell adhesion.

655A7-Pos Board # B571SCANNING OPTICAL FORCE IMAGING OF THEPLASMA MEMBRANE IN LIVE CULTURED CELLSUSING CD44 AS A PROBEKen Ritchie', Kotaro Koyasako2, Akihiro Kusumi'; lNagoyaUniversity/ERATO-JST, Chikusa-ku, Nagoya, 464-8602 Japan,2Nagoya University, Chikusa-ku, Nagoya, 464-8602 JapanRecent experimental work on single protein diffusion has shownthat most proteins diffuse in the plasma membrane through ahopping between small sub-micron domains. The membraneskeleton has been shown to play an important role in regulatingthis anomalous diffusion. To image the barriers to free diffusion inthe plasma membrane, a gold probe, sparsely coated with antibody,bound to the membrane protein CD44, (one that had beenpreviously undergoing diffusion) was trapped in an opticaltweezers and dragged in a raster scan across an area on a live NRKcell surface. Deviation in the probe's position from the center ofthe trap signifies a force applied on the gold-CD44 complex by theconstituents of the membrane. This measured force is used toimage the barriers to free movement. Networks of barriers with amesh size of about 0.5-1 Am were imaged that are presumably dueto the membrane skeleton. The force required to pass the barrierswas <0.5 pN and is dependent on the rate of scanning and thespring constant of the trap, as expected. Scans were performedwithin the following parameters: 2x2 pin area, scanning rate 0.5-2ju/s, trap stiffness 1-5 pN/gm.

655.48-Pos Board # B572A SINGLE MOLECULE STUDY OF A MULTIPLE-STEPRNA ENZYMATIC REACTIONXiaowei Zhuang', Harold Kim', Nils Walter2, Hazen Babcock',Steven Chu'; 'Stanford University, Stanford, Califormia 94305,2University of Michigan, Ann Arbor, Michigan 48109Complex biological reactions are often multi-step reactionsinvolving transient non-accumulative reaction intermediates thatare never significantly populated during the reaction course.Detecting these intermediates by ensemble methods is verydifficult, making the characterization of the kinetics of thesereactions a challenging task. It is thus desirable to study thesemulti-step reactions by single molecule methods that allow one tolook beyond the ensemble average, and measure time-trajectoriesof individual molecules. Using fluorescence resonance energy

transfer (FRET), we have studied the enzymatic reaction of thehairpin ribozyme at the single molecule level. The enzymecatalyzes the cleavage of a bound RNA substrate. It has beenproposed that the cleavage reaction happened in several steps: 1)substrate binding, 2) ribozyme folding to the active conformation,3) cleavage of the substrate, 4) conformation change of theribozyme to the inactive form, and 5) release of the cleavageproducts. A full characterization of this reaction by ensemblebiochemistry methods has proven to be difficult. In the singlemolecule FRET experiment, we have directly observed thetransient reaction intennediates and measured the rate constants ofindividual reaction steps. Our results strongly support the multi-step reaction model and provided a complete characterization ofthe reaction kinetics of this enzyme. This study shows that singlemolecule fluorescence is a powerful technique to characterize amulti-step biological reaction.

655A9-Pos Board # B573PEPTIDE SYNTHESIS BY SINGLE RIBOSOMESFrancesco Vanzil, Serguei Vladimirov2, Charlotte R Knudsen3,Barry S Cooperman2, Yale E Goldman'; University ofPennsylvania, Pennsylvania Muscle Institute, Philadelphia, PA19104, 2University of Pennsylvania, Department of Chemistry,Philadelphia, PA 19104, 3Department of Molecular and StructuralBiology, Aarhus University, Aarhus, DenmarkThe ribosome can be considered as a supra-molecular motor thattranslocates along an mRNA track. We have tested methods thathave been successful with classical motor proteins as mechanisticassays of ribosomal protein synthesis. Ribosomes are programmedwith poly-U and adsorbed to a mica surface. Upon addition of EF-Tu, EF-G, EF-Ts and 3H-Phe-tRNAR' in polymix buffer, theimmobilized ribosomes synthesize poly-3H-Phe at an average rateof -0.06 peptide bonds/min or >0.6 bonds/min after correction forthe presence of inactive ribosomes. This rate is compatible withmeasurements on suspensions of ribosomes under otherwisesimilar conditions. To study the motor activity of individualribosomes we attach latex beads (0.25 - 1.0 ;Lm dia.) to the 3'-biotinylated end of long poly-U (8 - 10 kbases). The range ofdiffusion limited by the mRNA tether, measured by fluorescencemicroscopy, decreases as active ribosomal translocation shortensthe tether. The velocity of active movement of single ribosomesalong the mRNA template is compatible with a peptide synthesisrate of -1.5 peptide bonds/min. Force can be applied to thetethered bead with laser tweezers. These experiments demonstrateprotein synthesis by individual ribosomes immobilized on amicroscope slide and show great promise for elucidating themechanism of ribosomal protein synthesis. Supported by the NIH.

655.5-Pos Board # B574OUTER HAIR CELL MOTILITY AS TWO-STATEPIEZOELECTRICITYKuni H. Iwasa; NIH, 9 Center Dr, Bethesda, Maryland 20892-0922Recent studies revealed that the motility of the outer hair cell isbased on a membrane motor, which directly uses electrical energy.This motility has been successfully described by "area motor"model, in which transfer of motor charge across the membrane isaccompanied by mechanical displacements of the membrane. This"area motor" is equivalent to a special class of piezoelectricity inwhich elemental transitions are between a small number of discretestates, presumably reflecting cooperative interactions in its protein-based structure. The discreteness of states introduces nonlinearity,which is exhibited as the voltage- and load dependencies in thepiezoelectric coefficient, in the membrane capacitance, and in themechanical compliance. A number of reciprocal relationships,which are expected for a piezoelectric material, are alsoexperimentally confirmed. The outer hair cell is characterized by arelatively good piezoelectric coupling constant up to 0.35 and anexceptionally high piezoelectric coefficient reaching 10 sC/N, at

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least thousand times larger than any reported man-made material.These contrasting values are primarily due to the large mechanicalcompliance of the cell.

655.51-Pos Board # B575ROTARY TORQUE OF STALLED F,-MOTORHliroyuld Noji, Hiroyasu Itoh, Kengo Adachi, Masasuke Yoshida,Kazuhiko Kinosita, Jr; CREST, Miyamae-ku, Kawasaki 216-0001JapanF,-ATPase is a rotary motor that rotates a central subunit fueled byATP hydrolysis. Its subunit composition is a313y6e. Three a andthree 1 subunits are arranged alternately like segments of anorange. Central y subunit penetrates the cavity of a3(3 ring. Each 1has a catalytic site and generates a rotary torque hydrolyzing ATPsequentially. This rotation is observed with an optical microscopeby attaching of long fluorescent probe, fluorescently labeled actinfilament, to y of F,-motor immobilized on glass surface. The rotarytorque is a slope of a rotary potential between catalytic 1 and y,therefore, a potential profile can be determined from a rotarytorque at each angle. To determine it, we attached a magnetic beadto y, and impose an external magnetic field to stall the rotationover 3600. From a difference of angle between a bead and externalmagnetic field, the stalled torque was measured at each angle, anda potential profile was estimated. Furthermore, a torque inpresence of an ATP-analog, AMP-PNP, or ADP was alsomeasured to study a change of the potential during ATPhydrolysis.

655.52-Pos Board # B576SIMULTANEOUS IMAGING OF ATP HYDROLYSIS ANDROTATION IN A SINGLE F,-ATPASE MOLECULETakayuM Nisbizaka', Kengo Adachi', Hiroyasu Itoh2, KazuhikoKinosita, Jr.3, Hiroyuki Noji', Kazuhiro Oiwae, Ryouhei Yasuda';'CREST "Genetic Programming" Team 13, 2Hamamatsu PhotonicsKK, 3Keio University, 'Kansai Advanced Research CenterF,-ATPase is a portion of ATP synthase and is an ATP-drivenrotary molecular motor made of single molecule. We visualizedindividual ATP turnovers in a single F, molecule immobilized on aglass surface using total internal reflection fluorescencemicroscopy. Rotation of the central shaft of F, was simultaneouslyvisualized in a bright-field image of small polystyrene beadsattached to the shaft. To observe rotation at a reasonable speed,both fluorescently labeled ATP and excess unlabeled ATP wereadded in the medium. Binding of a fluorescent ATP molecule to acatalytic site induced a stepwise rotation by 1200, and in mostcases, a second 1200 step was subsequently observed before therelease of the fluorescent ATP. The duration between the first andsecond steps decreased at higher concentrations of unlabeled ATP,indicating that the second step was induced by the binding ofunlabeled ATP to a second catalytic site. The release of thefluorescent AT? accompanied a third 1200 step, which was alsoinduced by binding of unlabeled ATP. Thus, the fluorescent ATPoccupied one of the three catalytic sites of F, for two step intervals(2400), and its release required the filling of the remaining twosites by unlabeled ATP. At least for the fluorescent ATP, which ismore sticky than unlabeled ATP, the F, motor appears to operate ina tri-site mode.

655.53-Pos Board # B577ROTARY MECHANISM OF F1-ATPASEKeno Adachi', Hiroyasu Itoh2, Takayuki Nishizaka', HiroyukiNoji, Ryohei Yasudal, Masasuke Yoshida3, Kazuhiko Kinosita,Jr.4; 'CREST, Nogawa, Kawasaki, 216-0001 Japan, 2HamamatsuPhotonics & CREST, Tokodai, Tsukuba, 300-2635 Japan,3T1TECH & CREST, Nagatsuta, Yokohama, 226-8503 Japan,4Keio University & CREST, Hiyoshi 3-14-1, Kohoku-ku,Yokohama, Kanawaga 223-8522The enzyme F,-ATPase has been shown to be a rotary steppermotor made of a single protein molecule. The mechanicalproperties of this motor have been elucidated by opticalmicroscopic imaging. Being driven by ATP hydrolysis at threecatalytic sites, the motor rotates, basically, in discrete 1200 steps.The motor can convert most of the free energy obtained by ATPhydrolysis to mechanical work. Much of the work is done whenATP is bound to the enzyme, and some at the release of ahydrolysis product(s). Hydrolysis itself does not produceappreciable work, and thus the role of hydrolysis is to reset themachine to an initial state. A small amount of work may be doneduring hydrolysis, which may be used to induce rotation in theproper direction. Reverse rotation of this motor is believed toresult in ATP synthesis from ADP and inorganic phosphate. Wediscuss possible mechanisms of this motor by comparing it with anelectrical motor of human design.

Imaging & Microscopy

656.01-Pos Board # B578TWO-PHOTON 3-D MAPPING OF TISSUE ENDOGENOUSFLUORESCENCE SPECIES BASED ON EXCITATIONAND EMISSION SPECTRALily Hsu', Peter D. Kaplan2, Tom M. Hancewicz2, Keith M.Berland3, Peter T.C. So'; 'M.I.T., 77 Massachusetts Ave., Room 3-335, Cambridge, MA 02139, 2Unilever Research, US Inc., 3EmoryUniversityDeep tissue imaging has important medical implications for thediagnosis of skin disease, wound healing, and tissue engineering.In order to study tissue physiology with microscopic resolution, weused two-photon microscopic imaging based on the excitation ofendogenous fluorophores. A number of fluorophores areresponsible for the autofluorescence observed. The identities anddistributions of these fluorophores have not been completelycharacterized. The different fluorescent species are expected tohave different fluorescence excitation and emission spectra.Principle component analysis can be applied to extractspectroscopic components from two-photon images. In ex vivohuman skin, we were able to acquire a five dimensional data set(3D space + excitation spectra + emission spectra). From this dataset, we extracted the major spectral components from these imagesand correlated these species with known tissue structures. Inaddition to providing insight into tissue physiology, we are able tooptimize the excitation wavelength for each biochemical speciesfor skin imaging applications. This work is supported by UnileverResearch, US Inc.

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656.02-Pos Board # B5793-D APPLE SKIN AUTOFLUORESCENCE STUDIED WITHTWO-PHOTON EXCITATION MICROSCOPYMartin J. vandeVen', C. J. deGrauw2, C. Huybrechts1, M.Ciscato', M. Sowinska3, F. Heisel3, H. C. Gerritsen2, T. Deckers4,M. Ameloot', R. Valcke'; 'Limburgs University Center,Universitaire Campus, Bldg. D, Diepenbeek, L B-3590 Belgium,2Utrecht University, Princetonplein 5, Utrecht, U. 3584 CCNetherlands, 3CNRS, Strasbourg, Cedex 2, 67037 France, 4RoyalResearch Station, Brede Akker 3, Sint-Truiden, L. B-3800BelgiumNon-destructive fluorescence imaging methods are beingdeveloped for rapid physiological screening of the complete skinsurface to detect storage diseases like bitterpit and storage scald.Fluorescence from Chlorophyll, NADH and flavin-nucleotides isused to elucidate the physiological state of the fruit. Earlier workshowed steady-state spectra of selected areas of apple skin(Sowinska et al., SPIE Proc., 3382, 100 1998). Now theautofluorescence of selected volumes of intact apple skin has beenstudied with two-photon excitation (TPE). Scans were located onthe equator and centered on the red and green colored side of allthe fruit. Apple varieties included Malus Domesticus Borkh. x,Granny Smith and Jonagold. The home-built microscope setup(Sytsma et al., J. Microsc. 191, 39 1998) was equipped with a 1.2NA, 60x, water objective; a 100 fsec, 80 MHz, Ti:Sa laser runningat 772 nm and a fiber coupled CCD camera for 2-D spectralscanning. Scan depths extended down to several tens of ltm overan area of 64 1sm2. Various structures show characteristiclifetimes between <1 and >5 nsec. Both fluorescence lifetimeresolved images (FLIM) and steady-state spectra are presented.Their physiological significance is discussed. This work issupported by the project "Fonds Slimme Regio" of the Province ofLimburg.

656.03-Pos Board # B580TWO PHOTON LASER SCANNING CONFOCALMICROSCOPY OF FM1-43 UPTAKE IN THE INTACTCOCHLEAClaudius B. Griesinger, Christopher David Richards, Jonathan FAshmore; University College London, Gower Street, London,WC1E 6BT United KingdomFast synaptic transmission in the mammalian cochlea occursbetween inner hair cells (IHCs) and auditory nerve fibers. Toinvestigate membrane recycling of IHCs we have applied theendocytosis marker FM1 -43 to the organ of Corti (OC) in an in situsystem. A small window was opened in the cochlear bone to allowaccess. Tight junctions were left intact and FM1-43 reached onlythe apical surfaces of OC cells. Imaging of cellular structures waspossible since 2photon confocal microscopy enables opticalpenetration of tissues. Superfusing the OC with FMI-43 (5 AM)selectively labelled IHCs and outer hair cells. A steady state signalwas achieved within approx 200s at hair cell apical sites. FM1-43was transported to the hair cell base within 300s, labellingperinuclear and cytoplasmic ER and "basal aggregates" close tosynaptic release sites. Apical endocytosis was dependent on theapical bath solution: endolymph(131 mMKV/ 23uM Ca++) reduceduptake rates.The rates were enhanced on elevating Ca++ to 1.5mMand still further on using perilymph (4.6 mM K+;1.5 mM Ca++).Endocytosis from basolateral membranes of hair cells was slowerand independent of external Ca'. These data require a multiplecompartment model of hair cells to explain apical and basalmembrane retrieval rates. Supported by MRC (UK) andBBSRC(UK).

656.04-Pos Board # B581A DETAILED CHARACTERIZATION OF TWO-PHOTONPOINT SPREAD UNCTION IN TURBID MEDIUMChen-Yuan Dong1, Eric Bevin1, Karsten Koenig2, Peter T. C. So';'Massachusetts Institute of Technology, Room 3-335, 77Massachusetts Ave., Cambridge, MA 02139, 2University of Jena,Jena, GermanyIn recent years, fluorescence microscopy based on multi-photonexcitation has become a major imaging technique for studying anumber of biological systems, at the cellular level. In the area ofdeep-tissue imaging, point-like excitation volume of two-photonmicroscopy allows excellent axial depth discrimination and thenear-infrared light source permits deep imaging of turbidbiological samples such as the skin. Nonetheless, image contrasteventually degrades as deeper structures in highly scatteringspecimen are imaged. In this work, we attempt to characterize thenature of such loss of image quality in depth. We will show adetailed characterization of the widths of two-photon point-spread-function (PSF). 0.1 gm fluorescent imbedded in gel samplescontaining scattering Liposyn Ill solution are imaged. Results fromboth an oil objective and a water objective at different depths willbe shown. In addition, we will show data imaging 4 gm redfluorescent beads imbedded in an uniform green fluorescent gelcontaining scattering Liposyn II. By analyzing the images in boththe red and green and red detection channels, we can characterizethe contribution of the PSF tail region in two-photon imagedegradation. Our study is aimed to understand the nature of two-photon image degradation in depth, which can be important foroptimize two-photon imaging in deep-tissue.

656.05-Pos Board # B582STRATEGIES FOR IMPROVING THE DEPTHPENETRATION OF TWO-PHOTON FLUORESCENCEEXCITATIONEmmanuel Beaurepaire, Martin Oheim, Jerome Mertz; ESPCI, 10rue Vauquelin, Paris, 75005 FranceOne of the principal advantages of two-photon excitationfluorescence (TPEF) microscopy over confocal microscopy is itsability to image deeper in turbid media while maintaining a highspatial resolution. The reasons for this are threefold: in TPEFniicroscopy 1) only ballistic excitation photons generatefluorescence, 2) ballistic and non-ballistic fluorescence photonsgenerate signal, and 3) the excitation wavelength is inherently lessscattering. We consider these points in detail. In particular, wepresent experimental results in which fluorescence excitation andcollection are examnined separately in turbid media. These resultsallow us to formulate and verify strategies for independentlyimproving the efficiencies of both excitation and collection, andthereby considerably improving the depth penetration of TPEF.

656.06-Pos Board # B583MULTIPHOTON IMAGING OF THE GLUTAMATERGICSIGNALING DYNAMICS IN THE SUPRACHIASMATICNUCLEUS.Liana R. Artinian', Weiming Yu2, Enrico Gratton3, Martha U.Gillettel; 'University of Illinois at Urbana-Champaign, Urbana,Illinois 61801, 2Northwestern University, 303 E. Chicago Ave.,Chicago, Illinois 60611, 3LFD, Urbana, Illinois 61801Circadian rhythms in manunals are generated and controlled by thesuprachiasmatic nucleus (SCN). Signals of solar cycles aretransmitted to the SCN via the monosynaptic retino-hypothalamictract that releases neurotransmitter glutamate to activate the SCNacceptor cells. In the SCN brain slice preparations glutamateapplication resets the clock with a phase delay early night andphase advance late night1. Early night glutamate induces a phasedelay via the release of the gaseous messenger nitric oxidel and,consequently, intracellular calcium release from ryanodine

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channels (CICR)2. We utilized the method of the multiphotonmicroscopy of the adult rat brain slice to visualize the effects ofglutamate on the key components of the glutamate-induced phasedelay signaling. Glutamate-induced calcium transients werevisualized with calcium fluorescence dyes, Calcium Green andFluo 4. Nitric oxide release was visualized with DAF fluorescencedye. The morphological organization of the SCN cells in the livingbrain slice was studied with the membrane dye Laurdan, and thesedata were used to estimate the size of the structures visualized withcalcium and NO fluorescence probes in the SCN. These novelapproaches to imaging the dynamics of the glutamatergic signalingcascade in living adult brain slices reveal new mechanistic insights.Supported by: NS33240 and NS35859.1. Ding, J. M. et al. Science 266, 1713-1717 (1994).2. Ding, J. M. et al. Nature 394, 381-384 (1998).

656.07-Pos Board # B584IMAGING SINGLE FLUORESCENCE LABELEDMOLECULES IN LIVING CELLS USING TWO PHOTONEXCITATION

Max Sonnleitner, Manuel A. Moertelmaier, Gerhard J. Schuetz,Hansgeorg Schindler; University of Linz, Altenberger Str. 69,Linz, 4040

Imaging the diffusion of single fluorescence labeled lipids by TwoPhoton Excitation (TPE) has been accomplished for an artificiallipid membrane. Here we show that single molecule detection withTPE is also possible on cells in vivo. This opens the perspective toutilize the advantages of TPE, e.g. the reduced background,increased lateral and axial resolution and the possibility ofsynchronous multicolor experiments, for single molecule imagingin living biological cells. As a first realization of this new

methodology we studied the lateral confinement of fluorescencelabeled lipid molecules to microdomains within the plasmamembrane. Utilizing the capability of TPE for simultaneousexcitation of different dyes, we studied the colocalization of lipidmicrodomains with various raft-specific proteins. For unambigousdiscrimination between fluorescence of dye molecules fromautofluorescence of cell compartments, a combination of a fullyautomated spectrometer with a highly sensitive CCD camera wasused. This setup allows for taking "true color images" of livingcells at the level of single fluorescence molecules.

supported by the Austrian Research Funds by Grants P12097-PHYand P12803-MED, and the Austrian Ministry of Science, GZ200.027/3-III2a/98.

656.08-Pos Board # B585A SYSTEM FOR OPTICAL RECORDING OF SINGLECHANNEL CURRENTS

Josef Kerimo, Heather A Clark, Boris Slepchenko, Leslie M loew;University of Connecticut Health Center, 263 Farmington Ave.,Farmington, CT 06030-1507The purpose of this work is to study single channel currents byoptical means. Here we demonstrate our experimental setup andthen use theoretical simulations to determine the feasibility of our

approach. Calcium current through single a-hemolysin pores in a

lipid bilayer was measured electrically. Concurrently, a calciumindicator dye was used to monitor the local fluorescence change.A wide-field fluorescence microscope was built for imaging thelocal fluorescence change and was integrated with the bilayerclamp apparatus. The microscope operates in either epi-illumination mode or in total internal reflection illumination modefor better background rejection. Fluorescence images werecollected simultaneously with the electrical recordings to followthe single channel dynamics. Three-dimensional simulations werealso done to study the spatiotemporal distribution of the

fluorescence. The simulations incorporated calcium buffering andbinding to the indicator, and diffusion in the solution. We lookedat several factors that can affect the outcome of the fluorescencesignal, but the steady state fluorescence signal from a single a-hemolysin pore is typically reached in less than one millisecondand is spatially localized to only a few microns in dimension.

656.09-Pos Board # B586IMAGING SINGLE MOLECULES IN SOLUTION NEAR AFUSED-SILICA INTERFACELloyd M. Davis', Wesley C. Parker', David A. Ball', John G.K.Williams2, Greg A. Bashford2, Daniel L. Grone2, Robert D.Eckles2, Lyle R. Middendorfl; 'University of Tennessee, B.H.Goerthert Pkwy, Tullahoma, TN 37388, 2LI-COR, Inc., 4308

Progressive Avenue, Lincola, NE 68504Single fluorescent molecules of unconjugated Bodipy-Texas Redin aqueous solution are imaged near a surface using total-internal-reflection excitation and through-sample collection offluorescence. A water immersion apochromat (N.A. 1.2, WD0.2mm) collects light from the sub-micron thick evanescent-fieldregion, a Raman-notch filter blocks elastic laser-scatter and a band-pass-filter blocks most Raman light scatter that originates from thesolvent. Inages obtained with a 5 MHz Pentamax Gen. IVintensified CCD and a 1 MHz Micromax back-illuminated CCDare compared and the relative merits of each camera are discussed.Molecules that momentarily or permanently stick on the surfacewithin the evanescent illumination zone are easily visible withlow-intensity laser illumination and camera exposure times of80-200 milliseconds. In order to acquire wide-field good-contrastimages of freely-diffusing molecules that have much shorterresidence times, the laser power is increased to 300 mW and thebeam passed through a mechanical shutter, which provides anintense 3-millisecond illumination synchronized to the cameraframe rate. While most molecules diffuse out of the evanescentzone before the next laser exposure, stationary or slowly movingmolecules persisting over several frames, and blinking of suchmolecules are occasionally observed.

656.1-Pos Board # B587OPTIMIZING THE DESIGN OF A MAGNETIC MICRO.MANIPULATION MICROSCOPE FOR CELLULAR ANDSINGLE MOLECULAR STUDIESHyuk-Sng Kwon', Chen-Yuan Doug', Jason D. B. Sutin2,Hayden Huang', Enrico Gratton2, Peter T. C. So '; 'MassachusettsInstitute of Technology, Room 3-335, 77 Massachusetts Ave.,Cambridge, MA 02139, 2Uniersity of Illinois at Urbana-ChampaignWe designed and optimized a magnetic micro-manipulatormicroscope intended for biological applications at the cellular andsingle molecular level. In our 8-pole design, samples mounted withmagnetic particles placed at the trap center can experience 3-Duniform force or torsional stress. The poles are designed andpositioned to allow a high numerical aperture, water immersionobjective (NA 0.9, Zeiss Achroplan) with 1.46 mm of workingdistance to be used. Combined with optimized wire winding andimplementation of back irons, magnetic field and gradient islocalized to the sample region. By placing the sample at the centerof the micro-manipulator, 3-D mechanical manipulation of thespecimen can be achieved. Our system is capable of exerting forceon the order of 800 pN per ferromagnetic particle 5 ltm indiameter. We will present data showing the 3-D force and torquecalibration. Biological applications of this magnetic micro-manipulation microscope will be discussed. In particular, 3-Dmechanical stress-strain response of cells and the effects of torqueon the binding of ligands to single DNA molecules will bediscussed.

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656.11-Pos Board # B588A NOVEL TECHNIQUE FOR THE SPECTRUM IMAGINGOF SINGLE FLUORESCENT MOLECULESYoshikazu Suzukd', Shinji Kamimura', Tomomi Tani2, KazuoSutoh'; 'University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo153-8902 Japan, 2Tokyo Metropolitan Institute of Medical ScienceSingle-molecule experiments provide information on biochemicalreactions or structural properties of individual macromolecules thatare usually asynchronous in ensemble experiments. Fluorescenceresonance energy transfer (FRET) has been a useful tool to detectinteractions between proteins and ligands, or conformationalchanges of macromolecules because spectrum change due to FRETis highly sensitive to the distance between two fluorescent dyes(donor and acceptor). For the single-molecule FRET, it is essentialto detect simultaneously at least two different colors offluorescence emitted from single fluorescent dyes. Here wepresent a novel technique to obtain fluorescence spectra fromsingle fluorophores with a direct-view dispersion prism placedimmediately under the objective. Combining this prism with thetotal internal reflection fluorescence microscopy, we could detectfluorescence of a single fluorophore (Rhodamine Red) as a video-rate spectrum image. Furthermore, we could also detect FRETsignal of single donor (Rhodamnine Red) and acceptor (CyS)conjugated to a single protein as a donor-acceptor spectrum image.This technique is expected to be a powerful tool for multicolorimaging of single fluorescent dyes.

656.12-Pos Board # B589IMAGING THE DYNAMIC EVENTS: FRET MICROSCOPYAmmasi Periasamy; University of Virginia, Biology, Gilmer Hall(039), Charlottesville, VA 22904Protein visualization using FRET imaging approach has asignificant advantage over methods such as X-ray diffraction,nuclear magnetic resonance, and electron microscopy. Digitizedvideo FRET and the laser scanning confocal FRET (C-FRET)imaging microscopy can reveal the spatial distribution of steadystate interactions between two protein partners in intact cells in 2-and 3-dimensions respectively. The use of fluorophores that areexcited by near UV light in these live cell-imaging studies islimited by problems associated with photobleaching andphotodamage. This limitation can be overcome throughapplication of the technique of two-photon FRET (2P-FRET),which uses red light to excite these near-UV fluorophores. Theinformation obtained using these methods regarding steady stateprotein interactions in intact cells, although valuable, falls short ofrevealing a key feature of the living system - its dynamicorganization. This degree of temporal resolution can be achievedusing the technique of fluorescence lifetime imaging (FLIM). TheFLIM method detects the nanosecond decay kinetics of afluorophore, and provides a spatial lifetime map for the probewithin the cell. The use of this technology will significantlyimprove and expand existing capabilities for studying the proteindynamic events inside the living cell.

656.13-Pos Board # B590SPECTRAL BLEED THROUGH AND PHOTOBLEACHING CORRECTION IN FRET MICROSCOPY.Masilamanei Elangovan, Ammasi Periasamy; University ofVirginia, W.M.Keck Center for Cellular hnaging,#39 Gilmer Hall,Dept. of Biology, University of Virginia, Charlottesville, VA22903Fluorescence Resonance Energy Transfer (FRET) imagingmicroscopy has been widely used to localize the proteinassociations in living specimen. In steady-state FRET microscopy,one can easily encounter the problems of bleed through signal fromone channel to the other, since the emission spectra of the donoroverlaps that of the acceptor and the excitation spectra of theacceptor overlaps that of the donor. So far, very little attention has

been given to spectral bleed through and photo bleachingcorrection of the labeled fluorophore molecules in FRETmicroscopy. In order to quantitate the FRET signal, we developeda methodology to correct the spectral bleed through andphotobleaching of donor (D) and acceptor (A) molecules fordigitized video FRET (DVFRET), confocal FRET (C-FRET), andtwo-photon excitation FRET (2P-FRET) microscopy. Wedemonstrated in this paper how one could correct the donor signalbleeding through to acceptor channel and vice versa for variousfluorophores, conventional and green fluorescent proteins (GFPs).We also implemented correction for the photobleaching.

656.14-Pos Board # B591A METHOD FOR DISTANCE MEASUREMENT BETWEENFLUORESCENT PARTICLES IN THE 10-200 NM RANGEKatarina Kis-Petikova, Enrico Gratton; University of Illinois atUrbana-Champaign, 1110 West Green Street, Urbana, Blinois61801We introduce a method to measure relative distances of fluorescentparticles of different color immobilized on a quartz surface in atwo-photon scanning fluorescence microscope, with two channelphoton counting detection. The method is sensitive in the 10-200nm range, filling the gap between fluorescence resonant energytransfer and far field light microscopy. Instead of raster scanningof an image, excitation beam is moved periodically in a circularorbit with a radius of the size of the point spread function (300nm), in order to achieve maximum sensitivity in the radialdirection. Fluorescence intensity varies periodically with thescanning frequency (500 Hz). Fast Fourier transform of thefluorescence signal during one or more orbit gives the modulationof the first hannonic, which depends upon the radial distance of theparticle from the center of scanning. The coordinates of the centerof mass of particles are calculated from the modulation and phase,simultaneously in the two channels and relative distance of theparticles is calculated. Accuracy of the distance measurement isdetermined by the total number of photons detected. Experimentsdemonstrating the advantages of the method were performed ongreen and red fluorescent spheres of different size.Supported by the NIH , PHS 5 P41 RR03155

656.15-Pos Board # B592ATOMIC FORCE MECHANICAL STIMULATION OF C.ELEGANS INDUCES ALTERATIONS IN SECONDHARMONIC SIGNALS WITH NEURAL RESPONSE ANDACTION POTENTIAL CHARACTERArtium Khatchatouriants, Alcxandra Manevich, Aaron Lewis,Leslie Loew, Millet Treinin; Hebrew University of JerusalemWe have integrated a fully functional scanned probe microscope,based on our research in near-field scanning optical microscopy[1], with a non-linear optical second hannonic microscope. Ourresearch over the last several years has demonstrated the highlysensitive nature of the second harmonic signal to membranepotential [2] and such membrane potential changes have beendetected even around single molecules [3]. Recently, the object ofour study has been the nematode C. elegans [4]. In this poster thesecond harmonic signal of behavioral mutants of this animal thatlack the ability to respond to mechanical stimulation are comparedwith nematodes that can respond to such excitation. These animalshave been stimulated with the on-line scanned probe microscopeand as a result, the simultaneously recorded second harmonicsignals excited with 60 femtosecond pulses at 780 nm, were alteredwith a time dependence that could be associated with neuralresponses and action potentials. The neural response like secondharmonic signal alteration has been detected with nanoNewtonstimulation and sub-millisecond time resolution. This approach ofintegrated microscopy appears to have considerable potential forprobing sensory processes associated with learned responses in thisanimal and a variety of other systems.

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1. K. Lieberman et al., Rev. Sci. Instr. 67, 3567 (1996); 2.Bouevitch et al., Biophys. J. 65, 672 (1993); 3. Peleg et al., Proc.Natl. Acad. Sci. USA 96, 6700 (1999); 4. Lewis et al., Chem.Phys. 245, 133 (1999).

656.16-Pos Board # B593SECOND HARMONIC GENERATION PROPERTIES OFFLUORESCENT POLYMER ENCAPSULATED GOLDNANOPARTICLES STUDIED BY HIGH RESOLUTIONNON-LINEAR OPTICAL MICROSCOPY.Paul Joseph Campagnola', Heather A. Clark', Joseph P.Wuskell', Leslie M Loew', Aaron Lewis2; 'University ofConnecticut Health Center, 263 Farmington Avenue, Farmington,CT 06030, 2Hebrew University, Jerusalem, Israel

We report on the synthesis and second harmonic generation (SHG)properties of a napthyl styryl chromophore (JPW4041) that ischemically linked to polymer coated 100 nm gold nanoparticles.The nanoparticles are coated with a styrene/methacrylic acidpolymer mixture and linked to the dye through a succinimidylester. This dye has a lipophilic tail and is readily incorporated intothe membranes of cells. We demonstrate that freely diffusing dye-conjugated nanoparticles in aqueous solution both produce largeSHG and two-photon excited fluorescence (TPEF) signals. Therelative efficiencies of the dye and the dye/nanoparticle conjugateswere determined by high-resolution laser scanning SHG imagingof live L1210 lymphocytes, where TPEF imaging was used tonormalize the observed SHG intensities. We find relative SHGenhancement of the conjugate compared to the unlinked styrylchromophore of approximately 20 fold and place a lower bound of100 fold relative to dye-conjugated latex beads. Fluorescencelifetime measurements indicate the observed signals are due togold enhancement rather than quenching. The photophysicalproperties of this chromophore are environmentally sensitive,suggesting the use of these labeled particles as second harmonicbased sensors.

656.17-Pos Board # B594HIGH-RESOLUTION FLUORESCENCE IMAGING BYHARMONIC EXCITATION LIGHT MICROSCOPYJan T. Frohn, Andreas Stemmer; ETH ZurichIn fluorescence microscopy, a lateral resolution beyond the wellknown Rayleigh criterion can be achieved by means of anonuniform illumination pattern containing high spatial frequencycomponents. The small light spot of scanning confocalmicroscopes is the most widely used example of such anonuniform pattern, theoretically resulting in a 1.4-fold improvedresolution. We show that in wide-field microscopy a harmonicexcitation pattern generated by interfering laser beams results in a100-nm optical resolution for objects emitting green light - a 1.5-fold improvement over confocal microscopy. We demonstrate thepotential of our harmonic excitation light microscopy by imagingmicrotubules in cultured cells and immuno-labelled histologicalsections of several different rat tissues. Furthermore, we presentresults from numerical simulations of the resolution enhancementachievable in all directions with a fully three-dimensionalharmonic excitation set-up.

656.18-Pos Board # B595BIOLOGICAL MEMBRANE IMAGING WITH MULTI-HARMONIC LIGHT MICROSCOPYLaurent Moreaux, Jerome Mertz; ESPCI-INSERM EP0002, 10rue Vauquelin, Paris, 75005 FranceMulti-harmonic generation (MHG) differs from multi-photonexcited fluorescence (MPEF) in that it involves coherent scatteringof light rather than incoherent radiative absorption and emission.We describe a rigorous theory of MHG specifically adapted to

microscopic imaging, and outline the differences and similaritiesbetween MHG and MPEF in terms of effective cross-sections,angular radiation patterns, total emitted power, spatial resolutions,etc. In particular MHG scales quadratically with the number ofscattering molecules and produces a radiation pattern that is highlysensitive to phase. We describe a versatile microscope whichprovides simultaneous and independent MHG and MPEF imaging,and present some applications of MHG microscopy which areinaccessible to fluorescence. These include the study of membranedynamics with sub-wavelength distance resolution, and the highlysensitive imaging of cell membrane potentials.

656.19-Pos Board # B596DONOR PHOTOBLEACHING FRET MICROSCOPY INCELL BIOLOGYDavid Dunlap, Raffaella Villa, Flavia Valtorta; San RaffaeleScientific Institute, via Olgettina 58, Milan, 20132 ItalyThe efficiency of fluorescence resonance energy transfer (FRET)can reveal contact between fluorochrome-labeled proteins inspecific cellular compartments when coupled with microscopy.This may be determined from paired conditions directly as the ratioof fluorescence intensities or lifetimes or indirectly as the ratio oftime constants of exponential decay of the fluorescence intensitydue to photolysis of donor fluorochromes. These methods avoidcorrections of either the registration of images recorded throughdifferent filters or the comparison of absolute fluorescenceintensities of donor and acceptor fluorochromes. For widespreaduse of this technique, standard specimens and an acuteunderstanding of the photo-physical processes involved are useful.With these aims, measurements of FRET between fluorophorespositioned along filamentous actin in adherent cells were madeusing fast, portable software routines. The photobleaching timeconstant data indicate that intersystem crossing does not determinethe rate of photobleaching. FRET efficiency reflected the spectralcoupling of the donor and acceptor fluorochromes as expectedfrom Forster theory, and microscopic, in situ measurementsrevealed efficiencies similar to those reported for actin in vitro.

656.2-Pos Board # B597INTEGRIN INTERACTIONS IN C. ELEGANS MUSCLESTUDIED IN VIVO BY FLUORESCENCE RESONANCEENERGY TRANSFER (FRET).Sophia Y. Breusegem, Nicholas P. Barry, A. Craig Mackinnon,Benjamin D. Williams, Robert M. Clegg; University of Illinois atUrbana-Champaign, 1110W. Green St., Urbana, IL 61801Fluorescence microscopy was used to study integrin interactions invivo in the nematode C. elegans. Integrins are importanttransmembrane receptor proteins involved in cell signaling andadhesion. In C. elegans muscle, dense bodies and M-lines anchorthe myofilament lattice to the cell membrane and the extracellularmatrix. These highly organized structures contain integrinheterodimers as well as cytoskeletal adapter proteins and signalingmolecules. They are analogous to the focal adhesion complexesfound in human cells and as such they are good systems toinvestigate integrin's adhesive and cell signaling functions.Fluorescence resonance energy transfer (FRET) between fusionproteins with GFP mutants of different color allows the detectionof direct intermolecular interactions in vivo. Transgenic animalswere created in which protein pairs in the dense bodies or M-linesare labeled with cyan fluorescent protein (CFP) as a donor andyellow fluorescent protein (YFP) as an acceptor. Several methodswere then used to detect and quantify the extent of energy transfer,including one- and two-photon fluorescence lifetime microscopy,ratio-imaging and photobleach-FRET. This research was supportedby NIH, PHS 5 P41 RRO3155.

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656.21-Pos Board # B598ANTIBODY-ANTIGEN ADHESION STUDIES USINGOPTICAL TWEEZERSSimone Kulin, Rani Kishore, Kristian Helmerson, JosephHubbard; National Institute of Standards and Technology,Gaithersburg, MD 20899-8424

Multivalent molecular adhesion processes play a key role in a widevariety of biological phenomena, such as immune response,infections, as well as cellular migration, communication, anddifferentiation. In many cases adhesion is mediated by interactionsbetween specific molecules via weak, non-covalent interactions.The strengths of these bonds fall into the range accessible withoptical forces found in optical tweezers. We investigate theadhesion of antigen DNP-lysine and the monoclonal antibody SPE-7 using a dual optical tweezers setup. The antibody and antigenmolecules are covalently attached to polystyrene microspheres andstochastic adhesion is monitored. The association and dissociationrates are determined for different surface densities of the specificreceptors. Interestingly, the detachment rate is independent of themaximum number of bonds that can be formed at any oneencounter. Using a theoretical description based on Fokker-Planckdynamics we can describe the detachment process as a renewalprocess in bond space. This model accurately describes themeasured rates and provides insight into the detachmentmechanism.

656.22-Pos Board # B599CALIBRATING AN OPTICAL TRAPHenrik Flyvbjerg', Kirstine Berg-S0rensen2; 'Riso NationalLaboratory, Postboks 49, Roskilde, DK-2000 Denmark, 2Nordita,Blegdamsvej 17, Copenhagen, DK-2100 Copenhagen 0 DenmarkThe force exerted by an optical trap on a dielectric bead in a liquidis optimally found from the power spectrum of the bead'sstochastic motion in the trap. We discuss some potential pitfallsof this approach, and give a procedure which avoids some sourcesof systematic errors. This procedure squeezes maximalinformation from data, while providing a check of theirconsistency.

656.23-Pos Board # B600TRAPPING FORCES, FORCE CONSTANTS ANDPOTENTIAL DEPTHS FOR DIELECTRIC SPHERES INTHE PRESENCE OF SPHERICAL ABERRATIONSAlexander Rohrbach, Ernst H.K. Stelzer; European MolecularBiology Laboratory (EMBL), Meyerhofstrasse 1, Heidelberg,60117 GermanyWe present and verify a theoretical model, which predicts trappingforces (escape forces), force constants (trap stiffnesses) andtrapping potential depths for dielectric spheres with diameterssmaller than or equal to the wavelength of the trapping light.Optical forces can be calculated for arbitrary incident lightdistributions with a two-component approach that determines thegradient and scattering force separately. We investigate theinfluence of spherical aberrations due to refractive index mismatchon the maximum trapping force, the force constant and thepotential depth of a trap, which are important for optical tweezersapplications. The relationships between the three parameters areexplained and studied for different degrees of spherical aberrationand various spheres (refractive indices n = 1.39..1.57, radii a =

0.lm .. 0.5gm, lambda = 1.064gm). We fnd that all threeparameters decrease with increasing distance to the coverslip.Effects that could make the interpretation of experimental resultsambiguous are simulated and explained. Computational results are

compared with experimental data found in the literature. A goodcoincidence can be established.

656.24-Pos Board # B601DETERMINING RELATIVE AND ABSOLUTE AXIALDISPLACEMENT IN OPTICAL TRAPSKeir C. Neuman, Elio Abbondanzieri, Thomas T. Perkins, StevenM. Block; Stanford University, Stanford University, Stanford, CAOptical traps permit precise measurement of pN-scale forces andnm-scale displacements in three dimensions (3D). Until recently,however, axial position information (along the optical axis, or z-direction) of trapped, microscopic objects has neither been asaccurate nor as accessible as corresponding information in thespecimen plane (the x- and y-directions). Recent developments inprecision, feedback-controlled 3D piezo stages facilitatecharacterization of optical traps in the z-dimension with nm-levelaccuracy. We present general techniques, based on experimentalmeasurements of forward-scattered light, for determining theheight of trapped beads relative to the coverglass surface, the z-displacement of a bead within the trap, and the ratio of axialmotion of the microscope objective to that of the trap center. Theheight of a particle is determined from the z-dependentinterference pattern of scattered light, which is well-described byMie scattering theory for the bead-coverglass system, andmatching the measured pattern with theory supplies the ratio ofobjective to trap motion. The relative axial displacement of thebead from the trap center can be quantified from the comparativelylarge change in total scattered intensity. Using these techniques,we have measured z trap stiffness with improved accuracy.

656.25-Pos Board # B602MAGNETIC TRAP MEASUREMENTS OF THREE-DIMENSIONAL CELLULAR DEFORMATION.Hayden Huang', Chen Dong', Hyuk-Sang Kwon', Jason Sutin2,Richard Lee3, Peter So'; 'Massachusetts Institute of Technology,2University of Illinois, Urbana-Champaign, 3Brigham andWomen's HospitalThe mechanical properties of cells have not been wellcharacterized despite the relevance to modeling cellular responses,especially to mechanical stresses. Studies that have beenperformed are often limited to two dimensions and fast responsetimes (less than a minute). Certain technologies such asmicropipette aspiration are limited by the amount of control overthe force being applied. We report the use of an octopolarmagnetic trap to study the response of cells undergoing a local,hundred-piconewton force applied over tens of minutes, with theforce transmitted via a magnetic bead attached to a cell. Bycoupling the trap with a two-photon microscope, data are taken intwo-dimensional slices that can be reconstructed to give a three-dimensional picture of deformation patterns. Results indicate thatthe cellular response can be either local or global, depending on thecharacteristics of the applied force, and the deformation is notlimited to the surface of the cell.

656.26-Pos Board # B603SIMULTANEOUS MEASUREMENT OF FLUORESCENCEAND MEMBRANE DYNAMICS IN CARDIAC MYOCYTES:COMBINED SCANNING ION CONDUCTANCE ANDLASER CONFOCAL MICROSCOPYA Shevchuk', J Gorelik', Y Gu', H Spohr', C Edwards', IVodyanoy2, M Lab', D Klenerman3, Y Korchevl; 'ImperialCollege, Du Cane Road, London, W12 ONN United Kingdom,2Naval Research International Field Office, London, NWI 5THUnited Kingdom, 3Cambridge University, Cambridge, CB2 lEWUnited KingdomWe report a novel method for simultaneous measurement ofmembrane and chemical changes in a localized region of a livingcell by combining scanning ion conductance microscopy withconfocal fluorescence microscopy. The technique allowsquantitative, high-resolution measurement of changes in cell heightat a particular point on the cell surface while retaining the cell

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functionality. A wide range of changes in cell height, from 10 to30000nm, can be measured with less than lmsec temporalresolution. Simultaneously we measure the local intracellular Ca2+or H+ concentration in the confocal volume directly under the cellsurface despite large cellular movement. Both cell contraction andfluorescence can be continuously assessed during relatively longexperiments. This is important when monitoring the cell responsesto different physiological stimuli. These results provide an insightinto the relationship between the local concentration of Ca2+ andH+ and the size of the local motion in contracting cardiacmyocytes.

656.27-Pos Board # B604FUNCTIONAL AND OPTICAL IMAGING OF LIVINGCELLS BY LOCAL APPLICATION VIA AMICROPIPETI'E.Y Negulyaevl, J Gorelik2, A Shevchuk2, A Rothery3, C Edwards2, IVodyanoy4, M Lab2, D Klenerman3, Y Korchev2; lInstitute ofCytology, St Petersburg, Russian Federation, 2lsnperial College,3Cambridge University, Cambridge, CB2 lEW United Kingdom,4Naval Research International Field Office, London, NW1 5THUnited KingdomScanning ion conductance microscopy (SICM) has wide potentialfor the imaging of living cells. The technique is non invasive andcapable of obtaining topographic resolution of 50 am. One majoradvantage of SICM over other scanning probe microscopy (SPM)methods is the micropipette used in SICM can also be used tolocally deliver reagents or light onto the cell surface. This can beused in conjunction with a patch clamp recording for functionalimaging or scanning near field optical microscopy (SNOM)respectively. We have mapped the distribution of active ionchannels in intact cell plasma membranes. The method combineswhole-cell patch clamp recording with concurrent SCIM imagingand highly localised stimulation of individual ion channels. Usingthis we have investigated the distribution of ATP-regulated K+channels (KATP channels) in cardiac myocytes. We found thatthese channels are not uniformly distributed but predominantlyorganised in small groups, which are aligned along the Z-groovesof the sarcolemma. The channels groups are anchored in thesarcolemma. They are separated by 2-6 micrometers and containup to 10 active channels. We have also combined SCIM andSNOM for the study the imaging of living cells. A particularfeature of the method is a reliable mechanism to control thedistance between the probe and the sample in physiological buffer.We demonstrate this new method by recording near field images ofliving cells.

656.28-Pos Board # B605LIVE CELL CARS (COHERENT ANTI-STOKES RAMANSCATTERING) MICROSCOPYAndreas Zumbusch, Thomas Hellerer; LMU Muenchen,Butenandtstr. 11, Muenchen, D-81377 GermanyFluorescence microscopic methods based on confocal microscopyor two photon excitation have become important tools in molecularbiology. Despite their value, problems inherent to fluorescenceexcitation persist. These include the phototoxicity of many dyes,their bleaching and not at least the need to overcome a strongbackground signal from sample autofluorescence. Recently wepresented a novel microscopic technique with contrast generationbased on Coherent Anti-Stokes Raman Scattering (CARS). CARSis a four-wave mixing process in which two laser beams interact togenerate a blue shifted signal. This process is resonantly enhancedif the frequency difference between the two laser beams coincideswith the frequency of a molecular vibration. It can be used tocreate a contrast for imaging with molecular vibrational selectivity.Due to the non-linearity of the process, three dimensional imagingwith a spatial resolution comparable to that of two photonmicrosopy is possible. Here we present a new setup that offersaccess to the whole vibrational spectral region. While the setup is

much simpler and easier to operate than the one used previously, itoffers a significantly improved signal to background ratio thereforemaking sub-minute image acquisition possible. The capabilities ofthe method are exemplified with live cell images.

656.29-Pos Board # B606NEW ADVANCES IN COHERENT ANTI-STOKES RAMANSCATTERING (CARS) MICROSCOPY ANDSPECTROSCOPY OF BIOLOGICAL SYSTEMS.Andreas Volkmer, Ji-xin Cheng, Lewis D. Book, X. Sunney Xie;Harvard University, 12 Oxford Street, Cambridge, MA 02138Coherent anti-Stokes Raman scattering microscopy allowsvibrational imaging and spectroscopy of biological systems withhigh sensitivity, noninvasiveness, and three-dimensional sectioningcapability [A. Zumbusch et al., PRL 82 (1999) 4142-4145]. Wehave recently made two significant new advances with respect tothe sensitivity of CARS detection, which was limited by thepresence of solvent and/or non-resonant signal. It is shown that anepi-detected CARS (E-CARS) geometry allows effective rejectionof the solvent signal, whereas time-resolved CARS detectionenables complete removal of non-resonant contributions. Bothmethods considerably improve the sensitivity of CARS imaging.Furthermore, we have extended the spectral region of CARSmicroscopy to 1000-1700 cm-l where protein and DNA signaturevibrational bands reside. Application to cellular imaging will bepresented.

656.3-Pos Board # B607CHEMILUMINESCENT DETECTION IN MICROARRAYPLATFORMS USING CCD IMAGING.Brady J. Cheek, Adam B. Steel, Hongjun Yang; Gene LogicIncorporated, 708 Quince Orchard Road, Gaithersburg, MD 20878Chemiluminescent imaging of microarray technologies for geneand protein expression is a highly desired alternative to fluorescentand radioisotopic methods of analysis. Chemiluminescence,however, has been seldom used in two-dimensional microarrayplatforms because adequate spatial resolution is difficult toachieve. In chemiluminescence, a non-emitting substrate isactivated to a freely diffusing, light emitting species by anenzymatic catalyst. The mobility of the activated substrate limitsthe spatial resolution that can be attained in an imaging system.We have developed an improved microarray platform in whichmolecular interactions occur within three-dimensional volumes ofordered microchannels rather than at a two-dimensional surface.The three-dimensional ordered microchannels localize the lightemitting species, and provide a unique system forchemiluminescent detection in a microarray platform. Luminolbased chemiluminescent reactions catalyzed by horseradishperoxidase were imaged and the analytical utility ofchemiluminescent imaging of gene expression patterns onmicroaffay platforms will be discussed.

65631-Pos Board # B608FLUCTUATION CORRELATION SPECTROSCOPY (FCS)IN TURBID MEDIA: DETECTION OF SOMATIC CELLSAND BACTERIA IN BODY FLUIDSAbdel Tahari, Guido Motolese2, Enrico Gratton'; 'University ofIllinois at Urbana-Champaign, 1110 W. Green St., Urbana, IL61810-3080, 2Pluristandard, Pechiera Borromeo, Milano, ItalyWe seek an alternate, simple, and cheap method to analyze cellsand bacteria in body fluids for clinical and biotechnologicalapplications; a task commonly performed using a flow cytometer, alarge, expensive instrument. FCS can detect and quantify singlemolecules in small volume. Single molecule sensitivity is obtainedwhen small volumes (=0.2 fL) of excitation is achieved. Bestsensitivity is when one molecule is contained in that volume, i.e.,for concentrations of -l OnM and the range spans =luM to =l OpM.Volume of observation can be increased for better sensitivity, but

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background fluorescence or impurities limit the lowest detectableconcentration. For analysis of body fluids, sensitivity on the orderof 100-100000 particle/ml is needed. Bacteria or somatic cellsstained with fluorescent dyes give large fluorescence signals, butvolume must be increased by =I0O to have about one particle in theobservation volume. Also, diffusion does not produce fast enoughfluctuations. We constructed a small, portable, easy to use, andrelatively inexpensive FCS instrument (designed to measuresomatic cells in milk) with high sensitivity and high dynamicrange. The sample is rotated at high speed to effectively scan alarge volume. Background signal is analyzed to provide accuratevalues of the number of cells in the physiological range. [Support:NIH, PHS P41 5 RR03155.]

656.32-Pos Board # B609A 3-) LASER SCANNING CYTOMETERKi Hean Kim, Karen Almeida, Carrie Hendricks, Molly Stitt,Bevin P Engleward, Peter T. C. So; Massachusetts Institute ofTechnology, 3-335,77 Mass Ave, Cambridge, MA 02139Flow cytometry is an important tool in cell biology where theproperties of a population of cells can be categorized based onmulti-parametric fluorescence measurements. Precision statisticscan be extracted from a cell population based on the device'sability to a large number of cells in a short time. While flowcytometry is a power technique, there are cellular properties, suchas cellular morphology and structure, that cannot be quantified byfluorescence spectroscopy alone. To address this need, 2-D imagecytometry has been develop where an automated microscopesystem has been used to image and categorize a population of cellsgrowing on a substrate. In this presentation, we present a 3-Dimage cytometer based on two-photon excitation which is a logicalprogression in developing the technology of image cytometry.Important biomedical questions can be addressed if the propertiesof cells inside intact tissues can be sampled. Based on two-photonexcitation, we have developed a 3-D image cytometer that iscapable of imaging into tissue up to a depth of 250 gm. Thissystem is capable of imaging up to a million cells in three hoursallowing important population statistics question to be addressed.The function of this 3-D image cytometer will be demonstrated inquantifying mixture of fibroblasts or embryonic stem cellsexpressing CFP and YFP at different concentration. We havedemonstrated the ability to quantify cells ratios as high as I in 105.This work is supported by NIH NCI.

65633-Pos Board # B610TOTAL INTERNAL REFLECTION WITHFLUORESCENCE CORRELATION SPECTROSCOPYTammy E. Starr, Alena M. Lieto, Randall C. Cush, Nancy L.Thompson; University of North Carolina, Campus Box 3290,Chapel Hill, NC 27599-3290Total internal reflection with fluorescence correlation spectroscopy(TIR-FCS) is a method for characterizing the dynamic behaviorand absolute concentrations of fluorescent molecules near theinterface of solution and a planar substrate. A general theoreticalform for the TIR-FCS autocorrelation function when both surfaceassociation/dissociation kinetics and diffusion through theevanescent wave contribute to the fluorescence fluctuations hasbeen derived. The autocorrelation function is predicted to depend,in general, on the kinetic association and dissociation rateconstants, the surface site density, the concentration of fluorescentmolecules in solution, the solution diffusion coefficient, and thedepth of the evanescent field. Approximate expressions applicableto samples in which either surface association/dissociation ordiffusion through the evanescent field is dominant have also beenderived. The theoretical results have been compared withexperimental TIR-FCS data for the diffusion of fluorescentlylabeled IgG through evanescent fields adjacent to substrate-supported planar membranes as well as for the specific interactionof fluorescently labeled IgG with Fc receptors that have been

purified and reconstituted into substrate-supported planarmembranes.

656.34-Pos Board # B611FLUORESCENCE LI#ETIME MEASUREMENTS INLIVING CELLS WITH A CONFOCAL LASER SCANNINGMICROSCOPE AND A STREAK CAMERA.Christoph Biskup,Thomas Zimmer, Klaus Benndorf; Friedrich-Schiller-University, Teichgraben 8, D 07740 Jena, GermanyFluorophores are widely used to study cellular structure andfunction. Generally, parameters of the fluorescence decay are notonly dependent on the molecular structure of the fluorophore itselfbut also on the microenvironment. Accordingly, emission spectrumand fluorescence lifetime of a fluorophore can provide importantinformation about its microenvironment.Therefore, it is attractiveto study these parameters in vivo in a small, confined volume. Weadapted a streak camera and a spectrograph to a confocal laserscanning microscope. This system takes benefit from both the highspatial resolution of the laser scanning microscope and the hightemporal resolution of the streak camera. By using this techniquewe measured in a small confocal volume both the emission spectraand the corresponding time domain based fluorescent lifetimes.For fluorescence excitation we used a femtosecond Ti:Sapphirelaser. A pulse picker was used to reduce the pulse repetition rate to2 MHz. To assess the performance of our system we measuredlifetimes for a variety of fluorophores. Our results show goodagreement with published non-confocal measurements. Currently,we use this method to measure lifetimes of GFP variants and toinvestigate interactions of GFP-labeled ion channel subunits.

656.35-Pos Board # B612DYNAMIC FLUORESCENCE DEPOLARIZATIONIMAGING MICROSCOPYAndrew Harry Clayton, Quentin S. Hanley, Donna J. Amdt-Jovin, Thomas M. Jovin; MPI fuir biophysikalische Chemie, AmFaBberg 11, Gdttingen, 37077 GermanyWe describe an extension of frequency domain fluorescencelifetime imaging microscopy (FLIM), denoted anisotropy-FLIM orrPLIM, which enables the measurement of rotational diffusion offluorophores on a pixel-by-pixel basis. The implementation ofrFIJM is achieved using existing wide field frequency-domainFLIM technology by introduction of linear polarizers in theexcitation and emission paths. Measurements of the phase delayand intensity ratio (DC and AC) between the polarized componentsof the fluorescence signal report on the fluorophore dynamics andare sensitive to the presence of heterogeneity in correlation times atthe molecular level. The technique and concepts of rFLIM areillustrated with model fluorophore-solvent systems and extended tolive cell measurements of acridine orange dynamics in 3T3 cells.Togetlher with 2-D image detection by a gain modulated intensifiedCCD camera, rFLIM provides wide-field imaging of dynamicdepolarization with simultaneous interrogation of differentcompartments of the cell. We discuss the interpretation of singlefrequency lifetime and rotational correlation data in aheterogeneous population of fluorophores.

656.36-Pos Board # B613BIOLOGICAL IMAGING USING STANDING WAVETOTAL INTERNAL REFLECTION MICROSCOPYPeter T. C. So, Jason J. Sutin, George Cragg; MassachusettsInstitute of Technology, Room 3-461, 77 Mass Ave, Cambridge,MA 02139Standing Wave Total Internal Reflection Microscopy (SW-TIRM)has the potential to provide lateral resolution on the order of 60 nmor better using 440 nm excitation light. This technique is based onthe formation of high frequency standing wave excitation in a totalinternal reflection geometry. Using a simple algorithm, high-resolution images can be re-constructed from a series of images at

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different phases of the standing wave. The ability to perform non-contact imaging of biological specimens at a resolution that iscomparable to or better than that of scanning probe microscopy isparticularly promising. This paper describes some biologicalapplications of this new technology. (Supported by NSF DBI9987122)

656.37-Pos Board # B614IMAGING BIOLOGICAL SAMPLES WITHPOLARIZATION-MODULATED DIC MICROSCOPY.David B. Hill, George M Holzwarth; Wake Forest University,Winston-Salem, NC 27109Differential interference contrast microscopy yields high-resolution images of unstained biological samples. Addingpolarization modulation to standard DIC with a computer-controllable liquid crystal variable retarder switches imagehighlights into shadows and vice versa in alternate frames. Adifference image is then computed and displayed at 30 frames/s byan image processor. The method automatically subtractsbackground. The resultant images have markedly improvedcontrast. Jones calculus analysis shows that, if the source intensityis adjusted to fill the camera wells, the image SNR is:SNR = sin(R)sin(D)/[(1-cos(R)cos(D))N]Here R is the phase shift introduced by the retarder, D is the phaseshift introduced by the sample, and N is the rms noise. Thisexpression has a maximum when R= D, which is confirmedexperimentally. Images of microtubules and rat brain slices, andmovies of epidermal keratocytes, will be shown. Details willappear in Applied Optics.Supported by NIH RR13358.

656.38-Pos Board # B615NEAR-FIELD FLUORESCENCE MICROSCOPY OFLIVING CELLS.Satoko Ohta-Iinol, Shun-ichi Ogawa2, Gerard Marriott3, AkihiroKusumi4; 'Nagoya University, Purocho, Nagoya, 464-8602 Japan,2ERATO, 5-11-33 Chiyoda, Nagoya, 464-0012 Japan, 3Universityof Wisconsin-Madison, 1300 University Ave., Madison, WI53706, 4Nagoya University/ERATO, JapanThe near-field scanning fluorescence microscopy of living cells, ata spatial resolution better than the diffraction limit (-100 nm), isdescribed. The near-field scanning optical microscope (NSOM)was constructed on an inverted microscope. Bent fiber tips coatedwith gold, with a -120 nm-q aperture, were used as probes. Weobserved cultured living epithelial cells that had been transfectedwith GFP-actin DNA. Using NSOM, we could observe fine anddark actin filaments that could not be observed by a conventionalfluorescence microscope. The movement of actin filaments couldbe imaged by repeatedly scanning the same field. The followingwere important for observation of living cells by NSOM: 1) readyswitching between epifluorescence and NSOM for quicklysearching and approaching with the NSOM probe to the cellstructure of interest, 2) easy operation for quickly setting thesample and the probe, which allows observation of cells while theyare still alive, and 3) tapping-mode scanning using a bent tipworking fully as an AFM probe, which allows scanning on the softcell surface without damage.

656.39-Pos Board # B616NEAR INFRA RED CONTRAST AGENT FOR TUMORCELLS EXPRESSING THE HIGH AFFINITY FOLATERECEPTORNicholas P Barry, Alison Bower, Sheela D Konda, Arun Singha,Erik C Wiener, Enrico C Gratton; university of illinois at urbanachampaign, 1110 w.green st., urbana, il 61801A number of tumors of epithelial origin including ovarian cancer,have been shown to express the high-affinity folate receptor, h-FR.

Optical detection of such tumors requires good penetration of lightinto the tissue of interest and some spectroscopic/ absorptivesignature to distinguish these cells from normal tissue. In this workwe investigate the use of folic acid conjugated to the NIRfluorescent dye, Indocyanine green as a tumor specific contrastagent.. This gives us active targeting of cells that express h-FR inaddition to good light penetration through tissue of both theexcitation light and fluorescence emission of the NIR dye. Withsufficient specificity of staining, this method may be used to verifyremoval of tumor cells during surgical procedures. We imageovarian tumor cells in a NIR laser scanning fluorescencemicroscope (excitation at 780nm). We demonstrate specificity ofbinding using in vitro grown ovarian cancer cell lines one positiveand one negative for expression of h-FR. In addition we assessnon-specific binding of indocyanine green alone and inhibition ofbinding in the presence of an antibody to h-FR. This workdemonstrates the potential use of this tumor contrast agent. Thiswork is supported by NIH PHS 5 P41-RR03155 PHS 5, P41RR05964-07, and I R29 CA61918.

656.4-Pos Board # B617INTERFEROMETRIC LIGHT SCATTERING STUDIES OFBRAIN ARCHITECTUREMark J. Schnitzer; Bell Laboratories, 600 Mountain Ave, MurrayHill, NJ 07974Traditional histological studies of the brain generally involve fixedtissue slices. However, studies of alert animals would greatlybenefit from methods for identifying brain laminae in vivo. Forexample, electrophysiologists often have difficulty placingelectrodes within known laminae, particularly in deep corticalareas with complex sulci and gyri. I am investigating the use oflow coherence interferometry (LCI) for studying infrared lightscattering signals within the living brain; such signals may helpidentify individual laminae. LCI is a heterodyne approach thatselectively detects miniscule reflections from a chosen depth belowthe tissue surface. Initial studies using a Ti:sapphire laser and ratbrain slices indicated that lamina specific scattering signals exist inhippocampus, cerebellum, and cortex. To recover such signals withLCL I constructed an instrument with over 90 dB of dynamic rangeand -25 micron resolution. This instrument incorporates a rapidscanning optical delay line that enables lkHz scans over severalmillimeters and provides independent control of heterodynefrequency, scan rate, and dispersion compensation. I have alsodesigned novel fiber optic lenses that can be directly inserted intothe brain. This system allows recovery of scattering signals over400 microns into tissue, and may ultimately enable identificationof laminae in vivo.

656.41-Pos Board # B618CULTURE SYSTEM FOR OBSERVATION OFINDIVIDUAL BACTERIAIppei Inoue', Yuichi Wakamoto', Kiyoshi Onuma', AkikoKashiwagi2, Kunihko Kaneko', Tetsuya Yomo2, Kenji Yasuda';'The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 Japan, 2OsakaUniversityTo investigate the interaction of bacteria with each other cells andwith their environment, we need to be able to continuously observesingle cells. We therefore developed an on-chip microculturesystem, a 7x7 array of chambers - each 30, 50, or 70 um indiameter and 10, 20, or 30 um deep is etched into a glass slide andcovered with a semipermeable membrane separating the chambersfrom the nutrient medium circulating through the "cover chamber".A single cell or a group of cells can thus be isolated from theothers perfused with same medium, the medium in each chambercan be changed within the diffusion time ( < ls ), and theinteraction between the specific cells or cell groups can becontrolled by using optical tweezers. We found that singleEscherichia coli enclosed alone in a chamber took more time tostart their first division than did cells in a chamber with others, that

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when cells were perfused with M9 medium the length of each cellincreased at 80 nm/min between cell divisions regardless of thechamber volume and the number of cells in it, and that the growthrate decreased when the nutrient concentration in the medium wasreduced. Effect of long-term isolation of single cells on theirgrowth and division will also be reported.

656.42-Pos Board # B619SPATIAL RESOLUTION OF HIGHLY OVERLAPPINGSPECTRAL COMPONENTS IN A FLUORESCENCE

IMAGING MICROSCOPE.

Joseph M. Beechem, Jason Kilgore, Walter K. Metcalf, RichardP. Haugland; Molecular Probes Inc., 4849 Pitchford Avenue,Eugene, Oregon 97402

A wide variety of multi-color fluorescence dyes have been

developed for fluorescence imaging. However, resolving all of the

spatial structures of interest within a single cell using only color

separation is difficult. Using fluorescence lifetime imagingmicroscopy (FLIM), one can spatially resolve overlappingemissions if they have different lifetimes. The major disadvantageof this approach, is the hardware expense associated with FLIM.We have been exploring alternate approaches, which exploitintrinsic differences in the "slow-time" behavior of spectrallyoverlapping dyes. For instance, fluorescein and Alexa Fluor 488have nearly overlapping spectra. However, fluorescein typicallyundergoes rapid photobleaching, whereas Alexa Fluor 488 is veryphotostable. By collecting multiple fluorescence images as afunction of time (seconds to minutes), one can effectively imagethe "bleachable" fluorescein green from the resistant Alexa Fluor488 green. Molecular Probes has a wide variety of fluorophoreswith very different "slow-time" photophysical characteristics. Asan example, we will show the resolution of 4-colors of greenwithin a single microscopic image. This "dye-centered" technologycan greatly extend the number of spatially resolved componentswhich can be simultaneously imaged.

656.43-Pos Board # B620

CORRELATION SPECTROSCOPY TOWARDSDETECTION OF ABNORMAL CELLS

Shih Chu Liaol, Beniamino Barbieri l, Weiming Yu2; 'ISS, 1602Newton Drive, Champaign, Illinois 61822, 2NorthwesternUniversity, 303 E. Chicago Ave., Chicago, Illinois 60611

Correlation spectroscopy performed using a fluorescencemicroscope, either with confocal or multi-photon excitation allowssensitive measurements of cellular activities in living cells. We are

using an integrated microscope system with correlation and cross-correlation data acquisition, and photon histogram and globalanalysis capability. The molecular mechanism involved in celldivision is intimately related to the fundamentals of understandingcancer. Signals, such as intracellular calcium level, during celldivision, is measured and analyzed. Due to rapid technologicaladvances and progress in new analysis methods, correlationspectroscopy is becoming an increasingly important tool forfinding abnormal signals, a 'new marker' for detecting abnormalcells.

656.44-Pos Board # B621

QUANTITATION OF HUMAN CAROTIDATHEROSCLEROTIC PLAQUE IN VIVO BY MRIJoel David Morrisett, Christof Karmonik, Christopher Eldridge,Wesley Vick, William Insull; Baylor College of Medicine,Methodist Hospital,A601;6565 Fannin St, Houston, Texas 77030Carotid atherosclerosis is a major cause of stroke in the westernworld. Interventions for the treatment of this condition includeaggressive lipid lowering therapy. Statins are now recognized notonly for their cholesterol lowering ability, but also for theirfavorable pleiotropic effects on the arterial wall. Non-invasiveassessment of this intervention is highly desirable, especially for

patients in whom serial measurements are required to monitoratherosclerotic plaque burden. We have used high resolution MRIand advanced image analysis to measure the plaque volume and %stenosis in contiguous 3mm slices of untreated Common (C1-C8)and Internal (I1-15) carotid arteries of 19 patients either before orafter endarterectomy. The mean plaque volume for slices C7 to C4was 30 mm3, rising to a maximum of 95 mm3at Cl adjacent to thebifurcation. Immediately above the bifurcation at II, the meanvolume was 50 mm3, decreasing gradually to 21 mm3 at I5. Incontrast, the plaque volume of the external branch was constant at19 mm3 from El to E4. Measurements of % area stenosisprovided complementary information. Slices C7 to C4 showedabout 18% stenosis, rising rapidly to 40% in slices C2 and Cl, thendecreasing to 30% in slices II to 13. This study illustrates thecapability of high resolution MRI for quantifying plaque burdenand % stenosis throughout the carotid arterial bed and formonitoring these parameters over time to evaluate medicalintervention for plaque regression.

656.45-Pos Board # B622CHARACTERIZATION OF ADENYLATE KINASE (AK1),ITS ISOFORM AKlB AND THEIR FUSING EGFPCONSTRUCT IN LIVING CELLQiaoqiao Ruan, Yan Chen, Michael Glaser, William W Mantulin;University of Illinois, 1110 W. Green St., Urbana, IL 61801Adenylate kinase (Myokinase E.C.2.7.4.3) is a ubiquitous enzymethat regulates the homeostasis of adenine nucleotides in the cell. Invertebrates, three isozymes have been characterized: AKI iscytoplasmic; AK2 and AK3 are localized in the intermembranespace of mitochondria. Recently, another AKI protein from murinecells was identified, namely AKIO (C. Schneider et al). Itssequence is identical to cytosolic AKI, except for an additional 18amino acids at the N-terminal. Our study showed that AKI frommurine cell when expressed in E. Coli, is competent to replace thebiological function of the native AKe. However, AKO1 can notreplace AKe's biological function in E.Coli. It is hypothesized thatthe 18 additional residues might serve as the signal and anchor formyristoylation. To better understand the function of these 18additional residues, the genes of AKl1I were fused with the gene ofEGFP (Enhanced Green Fluorescence Protein) and incorporatedinto an expression system. The construct was then transfected intoHela cells. Using two-photon microscopy imaging, we are able tovisualize the localization of AK1,B-EGFP inside the living Helacell. In addition, an AKI-EGFP construct was also introduced intoHela cells. The fluorescent images of AK1-EGFP and AKl,B-EGFP transfected cells showed distinct differences in proteinlocalization. The diffusion rates of these protein constructsmeasured by FCS (Fluctuation Correlation Spectroscopy)techniques reflect differences in local fluidity of the cell.(NIH, PHS 5 P41 RRO3155)

656.46-Pos Board # B623SYNTHESIS AND CHARACTERIZATION OF NEAR-IRVOLTAGE-SENSITIVE DYESReimund Engll, Mei-de Weil, Joseph P. Wuskell', Lawrence B.Cohen2, Michal Zochowski2, Leslie M. Loewl; 'University ofConnecticut Health Center, Farmington, CT 06030, 2YaleUniversity School of Medicine, New Haven, CT 06520The styryl and naphthylstyryl (ANEP) chromophores have provento be versatile foundations for the design of fluorescent fastvoltage-sensitive dyes. The existing dyes absorb in the 450-600nmrange and fluoresce in the 550-700nm range. Dyes with spectrafurther toward the red and near infrared would be advantageousbecause they would allow for minimal light scattering in thickpreparations, less photodynamic damage and lower backgroundfluorescence from intrinsic chromophores. We have synthesized aseries of over 20 new dyes with modified styryl chromophores thathave spectra ranging to 850nm. The incorporation of better donor

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and acceptor moieties at the opposite ends of the chromophoreprovided the most significant red shifts. The transmittance andfluorescence voltage responses have been determined as a functionof wavelength on a voltage-clamped hemispherical lipid bilayer.Several of the dyes have sensitivities as good as the best of theprevious styryl dyes. The signal to noise ratio on the lobsterwalking nerve was also assessed for each of the dyes. For example,JPW4023 has a furan linker to extend the chromophore withoptimal excitation and emission at 660 and 800mn, respectively; ithas a S:N twice as large as Di-4-ANEPPS, a widely usednaphthylstyryl dye. (supported by USPHS grants GM35063 andNS08437)

656.47-Pos Board # B624A NOVEL HIGH SIGNAL-TO-NOISE CA2+ PROBECOMPOSED OF A SINGLE GREEN FLUORESCENTPROTEIN.Junichi Nakai, Masamichi Ohkura, Keiji Imoto; NIPS, Myodaiji,Okazaki, Aichi 444-8585 JapanRecently, several groups developed green fluorescent protein(GFP)-based Ca2+ probes. When they were applied in vivo,however, they were difficult to use because of a low signal-to-noise ratio. Here we report the development of a high-affinityCa2+ probe composed of a single GFP (named G-CaMP). G-CaMP showed an apparent Kd for Ca2+ of 235 nM. Associationkinetics of Ca2+ binding were faster at higher Ca2+concentrations, with time constants decreasing from 230 ms at 0.2microM Ca2+ to 2.5 ms at I microM Ca2+. In HEK-293 cells andmouse myotubes expressing G-CaMP, application of drugs orelectrical stimulation induced fluorescent changes large enough tobe detected by naked eye. G-CaMP will be a useful tool forvisualizing intracellular Ca2+ in living cells. Mutational analysis,together with previous structural information, suggests the residueswhich may control the fluorescent intensity of GFP.

656.48-Pos Board # B625SOLUBILITY AND INTRACELLULAR DELIVERY OFHYDROPHOBIC VOLTAGE-SENSmIVE DYES WITHCHEMICALLY-MODIFED CYCLODEXTRINS.Andrew Bullen, Leslie M Loew; UCONN Health Ctr., 263Farmington Ave., Farmington, CT 06030The best voltage-sensitive dyes (VSD) are exceedinglyhydrophobic and are not easily loaded into cells. ComplexingVSDs with a water-soluble carrier molecule could conceivably aidintracellular delivery and staining. Cyclodextrins (CD) arenaturally occurring carbohydrate ring structures that are water-soluble but possess a hydrophobic binding pocket. The solubilityand inclusiveness of naturally occurring CDs can be increased bychemical modifications and the addition of hydroxyl acids (Redentiet al., 2000). Empirical determinations were made to determinewhich CD produced the best solublization and delivery of theVSD, di-8-ANEPPS. CDNSD complexes were loaded intocultured hippocampal neurons by dialysis through a patch pipette.In each case the relative solubility, staining efficacy (both intra &extracellular staining), background fluorescence and resultingsignal size was tested. Membrane potential dependent opticalsignals were elicited under voltage-clamp conditions and collectedusing a fast cooled CCD camera. All ,Bandy CDs could solubilizedi-8-ANEPPS to some extent. The resulting CDNSD complexeswere very water-soluble and could be easily loaded into cells toachieve intracellular staining. Both extracellular and intracellularstaining with CD/di-8 was improved resulting in an enhancedsignal to noise ratio. Additionally, the background fluorescence ofCDNSD complexes was decreased relative to the dye alone.(Supported by USPHS Grant GM35063)

656.49-Pos Board # B626BIDIMENSIONNAL CRYSTALLIZATION OF PROTEINS:MECHANICAL AND OPTICAL STUDIESSebastien COURTY, Francois GRANER, Jean-FrancoisLEGRAND; Universite Joseph Fourier, 140 avenue de laPhysique, St Martin d'Heres, 38402 FranceThe domains of applications extend from structural biology tobiosensors. The limiting step is the production of large highlyordered crystals. Today it is possible to grow bidimensional (2D)crystals of proteins at water surface if proteins adsorb, concentrateand self-assemble under a planar lipid film. In order to understandand possibly improve the mechanism of nucleation and growth of2D crystals at the air-water interface, we have developed anapproach combining optical and mechanical measurements. Theellipsometry enables to follow the process of protein adsorption onthe lipid surface but is not sensitive to the lateral crystalline order.This missing information is providing by the shear elasticmeasurement which allows us to monitor the kinetic of shearrigidity of the monolayer in real time. In complement, it appearedessential to us to image the surface during the formation ofcrystals, as well as during the shearing. Brewster AngleMicroscope (BAM), a non-destructive technique based on thesurface reflectivity properties, is useful to follow the first steps of2D crystallization. A static shear rigidity appears when thecrystalline domains percolate over the surface and keeps increasingfor several hours during a kind of annealing process of the 2Dcrystals.

656.5-Pos Board # B627MANIPULATION OF THE LOCAL STRUCTURE ANDCOMPOSITION OF MODEL MEMBRANESTerrence Gregory D'Onofrio, Christine Keating, Paul S. Weiss;The Pennsylvania State University, 152 Davey Laboratory,University Park, Pennsylvania 16802The physical properties and biological functions of membranes aredependent on the local structure of the lipid bilayer. Thedependence of the membrane curvature on the local structure andcomposition has been well documented. We hypothesize that thisrelationship is interdependent. We describe methods to modulatethe local structure of model membranes through control ofmembrane curvature. Our model membranes of choice are giantunilamellar vesicles prepared from multi-component lipidmixtures. We use lipid components known to phase segregate intodomains large enough to resolve with optical niicroscopy. Dual-color fluorescence microscopy techniques probe the local structureand enable us to map domain rearrangement as a function ofcurvature. Our methods to manipulate the curvature include"stretching" the membrane in the grasp of an optical trap, using abead as a "handle." Tension and corresponding mechanicalproperties may be measured with micropipette aspirationtechniques. We measure the local composition dependence ofthese mechanical properties, seeking to correlate local structurewith models of biological function.

656.51-Pos Board # B628LOCALIZED PHOTO-EXCITATION: MEASURINGDIFFUSION, MULTIPHOTON CROSS-SECION, ANDPHOTOBLEACHINGJ. Balaji, Parijat Sengupta, G. Ravindra Kumar, Sudipta Maiti;Tata Institute of Fundamental ResearchMultiphoton excitation (MPE) can non-invasively initiate micro-localized photochemical processes in biological systems. We havedeveloped an analytical theory for describing such three-dimensionally localized photochemistry, where the reaction iscoupled to diffusion in an open system. The analytical solutionsuggests a straightforward experimental method for measuringrelative diffusion constants. The power of this simple continuous

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photobleaching approach is demonstrated by measuring the changein the hydrodynamic radius of the protein barstar during unfolding.Optimal exploitation of this technique, and indeed of mostapplications of MPE in biology, requires quantitative knowledge ofthe absolute two photon absorption cross section (a2) of therelevant chromophores. We have solved this problem by obtainingan exact expression for the non-linear transmittance of a pulsedGaussian beam through a specimen of arbitrary thickness. Thispresents a simple but sensitive technique ("Far-field z-scan") formeasuring absolute 02. We have measured the 02 of tryptophan at532 nm with this technique, a measurement that requiredsensitivity more than an order of magnitude higher than thatdemonstrated by existing techniques. Par-field z-scan andcontinuous photobleaching together can measure the absolutevalues of the diffusion constant, two-photon cross-section and thequantum efficiency of photobleaching of a chromophore.

656.52-Pos Board # B629STATISTICAL ANALYSIS OF CELLULAR TRACTIONIMAGESWilliam A. Margansld, Micah Dembo; Boston University, 44Cummington Street, Boston, MA 02215Mechanical stresses exerted at the cell-substratum interface areimportant in many physiological processes such as wound healingand tissue formation. In order to quantify the stresses exerted on asubstratum by a cell, the elastic substratum method is utilized(Dembo et al., 1996). This method involves the dispersion ofmarker beads in an elastic substratum and the quantification of thebeads' displacements as the cell applies a traction field. Afterdividing the projected area of the cell into a mesh, the set ofdisplacement observations and a Bayesian statistical analysis areused to deduce the most likely traction field. In this study weimplement a Monte Carlo procedure to analyze how the accuracyof the predicted traction field depends on such factors as thenumber of marker beads and the error in the bead displacementmeasurements. Results indicate that the error in a traction field hasan inverse power law dependence on the number of beads. Thisrelationship holds for any given set of noisy bead displacementmeasurements. However, the error in a traction field does varylinearly with the error in the bead displacement measurements.Dembo, M., T. Oliver, A. Ishihara, and K. Jacobson. 1996.Imaging the Traction Stresses Exerted by Locomoting Cells withthe Elastic Substratum Method. Biophys. J. 70:2008-2022.

656.53-Pos Board # B630VISUAL SERVOING FOR THE DETECTION,QUANTITATION, AND MODULATION OF SPECIFICCELL RESPONSES IN SUBPOPULATIONS OFMULTIDRUG RESISTANT (MDR) HUMAN BREASTCANCER CELLS.Daniel E. Callahan, Bahram Parvin; Lawrence Berkeley NationalLaboratory, I Cyclotron Road, Berkeley, CA 94720MDR can be dependent on transmembrane proteins (e.g., PgP andMRP) that extrude foreign compounds. Expression of theseproteins is indicated by reduced accumulation of calcein. Anautomated digital imaging fluorescence microscope was used toperfuse calcein-AM (CAM, 0.2514M) containing modulating agentsMK-571 (10jiM) or verapamil (50AM) into a microperfusion cellchamber. MK-571 is a specific inhibitor of MRP, while verapamilinhibits both PgP and MRP. MCF-7ADR cells labeled withHoechst 33342 were observed at 35°C. Mean fluorescenceintensity per cell (MI, calcein) was calculated in real-time andsoftware operation of syringe pumps was based on thesecalculations; hence, the term visual servoing. The protocol foundin an MDR Beta-Test Kit (Molecular Probes, Eugene, OR), wasmodified. Images were acquired during perfusion intervals (1) 0-2100s, CAM, (Z) 8600-11000s, CAM+MK-571, and (i) 14000-16000s, CAM+verapamil. All cells responded by 3.

Subpopulations expressing MRP and/or PgP can be inferred basedon responses where AMI>200 (FIGI): (a) Responses in 1 (no PgPor MK-571) (b) None until 2 (MRP) (c) None in 1 or 2 (Pgp only)Funded by Department of Defense, DAMD17-98-1-8177 & U.S.Department ofEnergy DE-ACO3-76SF00098.

656.54-Pos Board # B631FLIFAST: SOFTWARE FOR FAST FLUORESCENCELIFETIME-RESOLVED IMAGE ACQUISITION WITHCONCURRENT ANALYSIS AND VISUAL FEEDBACKChristoph Gohlke, Oliver Holub, Robert M Clegg; University ofIllinois at Urbana-Champaign, 1110 West Green Street, Urbana, IL61801Fluorescence lifetime-resolved imaging FLI providesindispensable, valuable information than is not available fromsteady-state fluorescence image measurements. In general, becauseof the complexity of the analysis compared to the display of simpleintensity images, FLI measurements have required much longertimes to process and display. However, many biological andmedical applications using fluorescence imaging require real timeacquisition, processing and display. Real-time operation isnecessary for following dynamic biological events, or carrying outmedical diagnostics. We have developed a real-time FLI systemfor a variety of imaging applications. The instrument uses rapidfrequency-domain data acquisition hardware; however, here wedemonstrate software that specifically enables the real-timeprocessing and highly informative, convenient and easy-to-understand display of the lifetime-resolved fluorescenceinformation. A menu of possibilities is provided to the operator to

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assist in the on-line interpretation and control of real-timeexperiments and rapid events. The programs have been streamlinedin order to make the multiple-parameter, lifetime-resolvedinformation available to the user, and provide a continual up-to-date view of the statistics. NIH, PHS 5 P41 RRO3155.

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656.55-Pos Board # B632OPTICAL RECORDING: BACKPROPAGATION OFBURSTS OF ACTION POTENTIALS IN DENDRITES OFCULTURED HIPPOCAMPAL NEURONSBernd Kuhn, Peter Fromherz; Max-Planck-Institute forBiochemistry, Am Klopferspitz 18a, Martinsried, D-82152We examined the backpropagation of action potentials from thesoma into the dendrite of cultured hippocampal neurons of the rat.It is known from hippocampal slices that backpropagation during atrain of action potentials is attenuated.[1] We used a voltage-sensitive dye for our measurements.Dissociated neurons (ED 17) were cultured at very low density for8 to 37 days. The cells were stained with the novel voltage-sensitive dye ANNINE 5 [2] with a new 'HCl-method'.Measurements were done with a 90 x 60 pixel CCD-camera with aspatial resolution of 4.4 ;sm x 5.0 um and a time resolution of 0.75ms. In contrast to earlier measurements of backpropagation withphotodiodes [3], an area of 400 Am x 300 ,Am is observed withoutpiecing together and without averaging.We stimulated neurons at the soma for 800 ms, causing a train ofabout 15 action potentials. We observed the backpropagation of thefirst and the last action potential and compared both. There is onlya weak difference between them.Backpropagation in dendrites of cultured hippocampal neurons ismainly not activity-dependent, in contrast to hippocampal slices.

[1] N.Spruston, Y.Schiller, G.Stuart, B.Sakmann (1995) Science268, 297.[2] G.Htibener, P.Fromherz (unpublished).[3] E.Meyer, C.O.Mtller, P.Fromherz (1997), Eur. J. Neurosci.9,778.

656.56-Pos Board # B633TWO-PHOTON SINGLE PARTICLE TRACKING:INVESTIGATION INTO CHROMATIN MOVEMENT.Timothy Ragan', Andrew Belmont', Peter So2, Enrico Gratton1;'University of Illinois at Urbana-Champaign, lt10 W GreenStreet, Urbana, IL 61801, 2Massachusetts Institute of Technology,77 Massachusetts Avenue, Cambridge, MA 02139Recent research has indicated that the dynamics of chromatinmovement inside of the nucleus may be much quicker than whatwas previously thought. For example, large-scale chromatinstructure reorganization induced by transcriptional activation hasrecently been observed. In order to study chromatin in vivo weare using a single-particle tracking system that we have developedbased around the two-photon microscope. The system is capableof tracking particles by either of two methods: The first methodintroduces abefrations into the optical system which break the axialsymmetry of the point spread function and allows the 3Ddetermination of the particlej s position. The second method usesvolumetric scanning to localize the particle. The system has anextended axial range of 100 microns and a frequency response of15 Hz. To study the chromatin motion we have tagged regions ofthe chromosome by using lac repressor recognition of directrepeats of the lac operator with a GFP-repressor fusion protein.We present results on the motion of chromatin on the sub-secondtime scale and discuss the nature of the motion with respect toBrownian versus directed motion.

picosecond pulse trains. The vibrational contrast mechanismallows for imaging of live, unstained biological samples at anaverage power of only a few microwatts. Compared tofemtosecond pulses used previously, picosecond pulses allow twosignificant improvements for CARS microscopy: higher spectralresolution (-5 cmnl) and a larger ratio of resonant to non-resonantCARS signal. It is shown theoretically and experimentally thatepi-detection significantly improves sensitivity via effectiverejection of solvent signals. CARS images of living cells areobtained with high sensitivity, sub-diffraction spatial resolution,and the three-dimensional sectioning capability of this technique.These images vary based on the vibrational frequency that isprobed, which indicates that different cellular components can bedistinguished on the basis of their Raman spectra.

Theory I

656.58-Pos Board # B635MIRROR COMPETENCE IMPLIES PERSONCOMPETENCE.Otto E. Rossler, R. Rossler; Univ of Tubingen, 72076 Tubingen,FRGThe mirror competence (MC) of young chimpanzees is knownsince 1921 (Wolfgang Kohler) and 1969 (Gordon Gallup).Recently Daniel Povinelli questioned MC's sufficiency for a,,theory of mind". The human primate indeed has a further trait thatmay act as a catalyst. Happiness and bonding (laughter and smile)are virtually indistinguishable (J.A.R.A.M. van Hooff 1972). In her1978 ,,lnsights from the Blind", Selma Fraiberg showed thatchildren born blind (who see neither laughter nor smile) oftenremain ,,too harmless" for life outside an institution. ,,Smile-blindness" also often causes autism (lack of a theory of mind). Atherapy was discussed with the late Gregory Bateson in 1975:Acoustic mimicry of the optical condition (i.e., tender noises madeby the mother whenever she is happy). The same ,interactionalbifurcation" should then occur, via MC, as in the sighted: At first,the toddler starts to feed mother because he mistakes her laughterfor tenderness. When she feeds him next time, the exchangesymmetry causes him to experience ,,bene-volence". For the veryact just performed deliberately by himself feels good on thereceiving end. Next time, he tests the good effect over there and isrewarded by a careful return of the arrived joy. Mutualbenevolence is born in a positive feedback. Potentially benevolentagents are persons. Thus, personhood can be exported to smile-blind MC individuals. A ,,galactic export" is possible, including allMC species on the planet. They must, therefore, be granted theperson rights of Thomas Jefferson.We thank Rend Thomas and Jtirgen Neffe for stimulation. For

J.O.R

656.57-Pos Board # B634CARS MICROSCOPY OF LIVING CELLS WITH HIGHSPECTRAL RESOLUTION AND SENSITIVITYLewis D. Book, Ji-Xin Cheng, Andreas Volkmer, X. Sunney Xie;Harvard University, 12, Oxford Street, Cambridge, MA 02138A coherent anti-Stokes Raman scattering (CARS) microscope hasbeen developed that employs two synchronized Ti:sapphire laser

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