Effects of Glial Cells on Electrode Impedance Recorded from Neural

Prosthetic Devices In Vitro

JOHN P. FRAMPTON,1,4 MATTHEW R. HYND,2 MICHAEL L. SHULER,3 and WILLIAM SHAIN1,2

1Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY, USA; 2Wadsworth Center,New York State Department of Health, Albany, NY, USA; 3Department of Biomedical Sciences, Cornell University, Ithaca,

NY, USA; and 4Biggs Laboratory, Empire State Plaza, Albany, NY 12201, USA

(Received 13 July 2008; accepted 5 January 2010; published online 14 January 2010)

Associate Editor John A. White oversaw the review of this article.

Abstract—Neural prosthetic devices hold the potential to beused in the treatment of a variety of neurological disorders.However, their long-term clinical success is currently limitedby the ability to achieve stable interfaces between devices andthe CNS. Immunohistochemical analysis has shown thatcellular responses occur in tissue surrounding implanteddevices. These cellular responses have been correlated withthe impedance measured from device electrodes, leading tothe hypothesis that a possible mechanism resulting ininconsistent device performance is the formation of anelectrically insulating glial sheath at the implantation site.However, little is known about what cellular and tissuechanges affect impedance values and thus contribute to thedecreases in electrode performance. We have designed anin vitro system in which cell conditions can be varied withinan artificial tissue matrix surrounding a neural prostheticdevice. In this study, high-density cultures of glial cells wereanalyzed by immunohistochemical methods and impedancespectroscopy. Astrocytes and microglia were cultured atvarious ratios within the matrix surrounding the probes, andwere observed over a period of 2 weeks. Cell seedingconditions and confocal images were compared to impedancedata to enable the effects of glial cell type on electrodeimpedance to be determined.

Keywords—Reactive responses, Astrocyte, Microglia, Algi-

nate, Three-dimensional, Cell culture.

INTRODUCTION

Neuroprosthetic devices hold the potential torestore function to regions of the central nervous systemdamaged by injury or disease.9,27,35 Clinical applica-tions for such devices include stimulation of deep brain

areas for treatment of Parkinson’s disease,23,37 epi-lepsy,1 and traumatic brain injury,33 as well as non-invasive cortical stimulation and recording used insome brain computer interfaces.32,43 Neuroprostheticdevices have been fabricated with the capability to notonly stimulate and record signals from populations ofneurons via on-board circuitry, but to also reduce tis-sue reactivity by delivering neurotrophins or thera-peutic agents through microfluidic channels.30,38,42

Even so, long-term clinical applications are currentlylimited by the capability to form stable interfacesbetween devices and the central nervous system and theability to control unwanted cellular and tissue reactiveresponses around devices.8,28

The changes in tissue that occur in response toimplantation of neuroprosthetic devices have beencharacterized in animal models.4,18,39 Damage causedby implantation is similar to other types of traumaticbrain injury and can lead to changes in gene expres-sion,13 increased cell proliferation,10,22 compromisedblood–brain barrier integrity,29 infiltration of circu-lating macrophages, intermittent ischemia, and gener-ation of reactive oxygen species and inflammatorycytokines.34 Brain responses occur not only as a resultof the immediate damage caused by implantation, butalso in response to the sustained presence of the device.The sustained responses consist of changes in glial cellvolume, as well as migration to and proliferation ofactivated glial cells at the device–tissue interface.39,40

Over time, the activated cells can surround the devicewith an electrically insulating glial sheath. The glialsheath consists of a dense network of glial cell bodiesand processes that electrically insulate device elec-trodes, thereby preventing recording and stimulationof neurons; the result is inconsistent/unreliable deviceperformance.18,24

Address correspondence to John P. Frampton, Biggs Laboratory,

Empire State Plaza, Albany, NY 12201, USA. Electronic mail:

jf7674@albany.edu

Annals of Biomedical Engineering, Vol. 38, No. 3, March 2010 (� 2010) pp. 1031–1047

DOI: 10.1007/s10439-010-9911-y

0090-6964/10/0300-1031/0 � 2010 Biomedical Engineering Society

1031

While the glial sheath has been studied byimmunohistochemical and microscopic analysis offixed animal tissue,38–40 it has been difficult tocharacterize the brain responses in living tissue, andin real time. Impedance spectroscopy is one methodthat can be employed for real time analysis of reac-tive responses. Impedance spectroscopy measures theflow of current between electrodes in an electrolytesolution, allowing the resistive and capacitive circuitelements of the electrodes, and the medium betweenthe electrodes, to be deduced.3,17 This technique wasused in live rats to monitor the resistance andcapacitance of the brain tissue surroundingimplanted devices.41 Increases in tissue impedancewere correlated with increases in astrocyte reactivityobserved by microscopic analysis of tissue labeled forglial fibrillary acidic protein (GFAP). Impedancespectroscopy has also been used to monitor theaffects of cell type on electrode performance in 2Din vitro cell culture systems.26 These techniques havebeen used to monitor the electrical properties of thedevice–tissue interface. However, the contributionsthat individual cell or tissue variables have on tissueimpedance are still unclear.

We have constructed a 3D in vitro system that canbe used to assess individual components of the reac-tive response, and determine how they affect changesin the recorded impedance spectra. Our system con-sists of a microfabricated neural probe containingon-board electrodes, surrounded by a functionalizedalginate hydrogel matrix capable of holding neuralcells in three dimensions (3D). This system was pre-viously characterized in vitro using an astroglia cellline.16 Changes in cell density within the matrix sur-rounding the probe produced changes in the recordedimpedance spectra.16 As glial cell density approachedthe density characteristic of cortical tissue, theimpedance spectra shifted relative to the bare elec-trode, as a result of changes in the resistance andcapacitance of the glial tissue construct surroundingthe device.

In the present study, we have investigated the effectsof specific cell types and length of time in culture onimpedance spectra recorded from neural probes. Wehypothesized that changes in impedance spectra evolveover time in culture, and that different cell types causechanges in the impedance spectra consistent with thosecells’ morphology and function. To confirm this, weentrapped both microglia and astrocytes in alginatescaffolds around neural probes, at various ratios, usingtotal cell densities similar to those observed in vivo.Impedance spectra were recorded from electrodes at 3,7, and 14 days in vitro, and were subsequently inter-preted through correlative immunohistochemical andconfocal microscopic experiments.

METHODS

Cell Culture

Cultures of primary glial cells were established fromlitters of postnatal-day three rats.2 All procedures wereapproved by the Wadsworth Center InstitutionalAnimal Care and Use Committee (IACUC). Postnatal-day three Sprague–Dawley rats (Taconic Farms,Germantown, NY) were decapitated, the skulls werecarefully cut away, and the brains were removed andplaced in ice cold balanced saline solution (BSS) con-sisting of Ca2+- and Mg2+-free Hanks solution withadded antibiotic–antimycotic solution per manufac-turer’s instructions (Invitrogen, Carlsbad, CA). Brainswere prepared for microdissection by separation of thehemispheres and removal of the midbrain and cere-bellum, using fine surgical scissors. Cortical hemi-spheres were retained, but the meninges were removedusing hooked forceps. The hippocampal formationswere then dissected away from the cortices usingmicrodissection scissors and fine tipped jewelers’ for-ceps. Cortices were minced into 1 mm3 pieces using ascalpel, and centrifuged at 45 rcf for 5 min to form atissue pellet. Cortical tissue was dissociated by enzy-matic digestion in BSS containing 0.25% trypsin and50 lg/mL deoxyribonuclease (both from Sigma,St. Louis, MO). Complete dissociation was confirmedby trituration of the cell suspension 10–15 timesthrough a Pasteur pipette and filtration of the sus-pension through an 80-lm pore-size nylon mesh(Wildlife Supply Company, Buffalo, NY). The enzy-matic reaction was quenched by addition of an equalvolume of serum-containing medium. Cells were cen-trifuged at 250 rcf and plated on 100 mm2 Petri dishestreated for tissue culture (approximately one dish perbrain). Medium was changed once 24 h after plating,and every 7 days thereafter.

At approximately 5 days in culture, microglia beganto appear in the glial cultures. These cells were distin-guished based on cell morphology and on reactivitywith antibodies recognizing the microglia-specificmarker Iba-1. Microglia were found to loosely attachto both the surface of astrocytes, and the culture dish.Microglia were subcultured from primary mixed glialcultures as follows. Mixed glial cultures were incubatedin serum-free HEPES-buffered Hanks’ Saline (HBHS)for 15 min and then aspirated repeatedly with a 10-mLFalcon pipette. The HBHS containing detachedmicroglia was centrifuged at 250 rcf for 5 min, andthen cells were resuspended in culture medium, andplated on separate Petri dishes. Dulbecco’s modifiedEagle’s medium containing 10% FBS and 1 mg/mLprimocin was used as the culture medium for allexperiments. Glial cultures were passaged a maximum

FRAMPTON et al.1032

of three times and were maintained at 37 �C and5% CO2.

Alginate Modification and Creation of Tissue Scaffolds

Alginic acid sodium salt was obtained from Sigma.Sigma alginate is composed primarily of D-mannuronicacid residues containing minimal impurities from themanufacturing process. Numerous reports have char-acterized the physical properties of alginate (e.g. poresize, diffusion/permeability and substrate mechanics)and demonstrated its suitability as a mammalian cellculture matrix.11,12,14,20,21,31 Alginate was dissolved inHBHS at 10 mg/mL and dialyzed against MilliQ-H2O(18.2 MX) for 48 h in a 3500 molecular weight cutoffSnakeskin dialysis membrane (Pierce, Rockford, IL) toremove trace amounts of impurities. Alginate wasfunctionalized to promote cell attachment as describedpreviously,31 using GGGGRGDY (Wadsworth CenterPeptide Synthesis Core) to promote attachment of glialcells. Substrates, including probes and glass, wereprepared for scaffold attachment by incubation inpoly-L-lysine (PLL) (Sigma) for 1 h, followed by threeconsecutive washes in MilliQ-H2O, and air drying.Alginate scaffolds were created around neural probes(NeuroNexus Technologies, Ann Arbor, MI) byapplication of 20 lL of peptide-modified alginatesolution to the probe surface. Cells were included inthe alginate solution as the experimental designrequired. Crosslinking of alginate into a column of gelwas achieved by compression of the alginate solutionunder a 0.4-lm pore-size MilliCell tissue culture insert(Millipore, Billerica, MD) containing 200 mM buf-fered CaCl2 solution in the chamber of the insert. After30 s the insert was removed, and the scaffold waswashed three times in HBHS to remove excess Ca2+,before medium was placed on the cultures. The algi-nate constructs were up to 100 lm thick and coveredthe entire length of the probes (Supplementary Fig. 1).

Cell Seeding Conditions

Cell ratios of 100:0, 50:50, 25:75, 12.5:87.5, and0:100 microglia:astrocytes were tested for impedancechanges within 3D alginate constructs. Microglial werecollected as described above from both microgliasubcultures and mixed glia cultures. Astrocytes werecollected by washing glial cultures in HBHS andincubating for 5 min in TrypLE (Invitrogen). Separatepopulations of microglia and astrocytes were resus-pended in HBHS and counted with a hemacytometer.The total cell numbers of each cell type were deter-mined. The appropriate volume of each cell suspensionwas placed into 50-mL Falcon tubes, centrifuged, and

resuspended in a defined volume of alginate solution toprovide sufficient cells to meet the required cell densityand microglia:astrocyte ratio after centrifugation andresuspension in alginate solution. The alginate cellsuspensions were then crosslinked around the probesas described. Cell cultures were maintained up to14 days at 37 �C and 5% CO2.

Impedance Measurements

Microfabricated neural probes containing 16 irid-ium electrodes (177 lm2 surface area/electrode, spaced100 lm center-to-center) were mounted on custom-built glass frames fitted with Teflon rings for holdingmedium.16 Impedance measurements were made inHBHS with a Gamry potentiostat (Gamry, Warmin-ster, PA) at frequencies ranging from 10 Hz to 10 kHz,50 mV AC. Prior to application of cells, the impedanceof the bare electrodes was recorded in HBHS.Impedance spectra were subsequently recorded at 3, 7,and 14 days after application of glial cells. Beforeimpedance was measured, culture medium wasremoved, and cultures were rinsed twice, 1 min eachtime in HBHS. Impedance was measured from deviceelectrodes by way of a custom-made adaptor con-necting the probe printed circuit board (PCB) to theworking lead of the potentiostat. An Ag/AgCl elec-trode (Gamry) served as the reference electrode, and aplatinum foil served as the counter electrode. Refer-ence and counter electrodes were located approxi-mately 10 mm above the alginate constructs. Deviceelectrodes with abnormally large bare electrodeimpedances (gigaX at 100–10 Hz) or discontinuousphase spectra were excluded from analysis. Impedancespectra were initially analyzed in Gamry Frameworkand Echem Analyst software (Gamry). For graphingand statistical analysis, necessary data were exportedfrom Echem Analyst. To recycle devices after spec-troscopic analysis and imaging, electrodes werecleaned for five min in 20 mM EDTA to cause disso-lution of alginate which was carefully removed fromthe probe site using a pipette tip attached to a vacuumline. Probes were washed sequentially in continuousstreams of water, acetone, isopropanol and ethanol,using brief applications from a squirt bottle. Everyeffort was made to avoid exposure of the PCB to sol-vents. Devices were dried under a stream of nitrogengas and plasma cleaned for three cycles under airplasma.

Immunohistochemistry

Samples were fixed for 15 min in 4% paraformal-dehyde buffered in 25 mM PIPES, 10 mM HEPES,2.5 mM MgCl2, pH 7.4, and were then permeabilized

Effect of Glial Cells on Neural Prosthetic Impedance 1033

in 0.1% Triton X-100 for 5 min. Samples were washedthree times, blocked in 5% bovine serum albumin(BSA) for 30 min, and incubated in primary antibodiesraised against GFAP (mouse 1:1000) and Iba-1 (rabbit1:1000), to label astrocytes and microglia, respectively.Samples were blocked again in 5% BSA for 30 minand incubated in secondary antibodies (Alexa594 goatanti-mouse and Alexa488 goat anti-rabbit, each1:1000). All antibody incubations were performed onan orbital shaker overnight at 4 �C. After immunola-beling, samples were incubated in ribonuclease (10 mg/mL) for 3 h at 37 �C to degrade cytoplasmic RNA,and washed 3 times, for 30 min each, in HBHS prior tolabeling nuclei with TOTO-3 nucleic acid stain (1:500)at room temperature for 1 h. This step was necessaryto reduce cytoplasmic signals from TOTO-3 labeling ofRNA. Samples were rinsed three times for 5 min inHBHS between successive labeling steps, unlessotherwise noted.

Microscopy

Samples were prepared for microscopy as wholemounts of tissue constructs surrounding the probes.Approximately 10 lL of glycerol based mountingmedium containing n-propyl gallate was applied to thesurfaces of the constructs, and a 25 mm2 glass cover-slip (0.17 mm thick) was carefully placed on top. Thesamples were then inverted and placed on a manuallycontrolled microscope stage, using a stack of glasscoverslips as shims to balance the probe setup withrespect to the optical plane, thereby minimizingreflections. Confocal data sets were collected on aLeica SP5 confocal system using a 209 air objective(NA 0.7) and a 29 optical zoom (Leica, Wetzlar,Germany). Images were collected from the surface ofthe alginate cell constructs down to the optical plane ofthe neural probe (approximately 50 lm). Data setswere collected as a series of optical sections using0.384 lm steps. The final image data set dimensionswere 387.5 lm 9 387.5 lm 9 ~50 lm, x–y–z). Con-structs were not imaged below the level of the probe,since only the regions of the constructs above theelectrodes were expected to contribute impedance andbecause the probe interfered with data collection belowthis plane. Channels were collected sequentially, so asto minimize spectral overlap; the 488 (Iba-1 signal) and647 (TOTO-3 signal) channels were collected in thefirst scan, and the 594 (GFAP) channel was collected inthe second scan.

Data Analysis

Experimental impedance data were normalize todata for bare electrodes for several reasons. First,

previous results demonstrated that alginate alone hadno effects on impedance.16 Second, cell-free and cell-containing measurements would have to be madesequentially, e.g. after recycling of a device. There is noguarantee that the impedance properties of an indi-vidual electrode would be the same for both mea-surements. Impedance spectra were plotted asmeans ± SEM using Sigmaplot with statistical analy-ses performed using the Systat program. Shaded datamatrices were plotted to illustrate relative change fromthe bare electrode for both impedance modulus andphase. T-tests were performed using Systat to deter-mine statistical significance between culture conditions.A minimum of three devices (approximately 16 elec-trodes per device; total of up to 48 electrodes) wereanalyzed for each cell seeding condition. For inter-pretation of spectral changes, EIS data were analyzedin Sigmaplot and Systat to apply regression models.Data were processed in Sigmaplot to describe statisti-cal differences between measurement groups withoutforming any a priori assumptions about the data.Impedance modulus and phase were fitted separatelyusing either an inverse first order polynomial function(f = Y0 + (a/x); impedance modulus) or a quadraticfunction (f = Y0 + ax + bx2; phase angle).

Confocal images were imported into ImageJ asLeica image files. Image sequences were projected asmaximum intensity in the x–y and x–z planes.Approximate probe and electrode positions weresuperimposed onto the maximum intensity projectionsby tracing the autofluorescence signals from the probesand iridium electrodes observed in a single opticalsection containing this information. Adobe Illustratorwas used to trace the probe.

RESULTS

Characterization of Baseline Impedance

The impedance for 263 bare electrodes was mea-sured from glial cell cultures. Bare electrodes hadimpedance profiles characterized by a linear increase inimpedance magnitude as frequency decreased. Theaverage impedance magnitude ranged from 61.5 kX at100 kHz to 150 MX at 10 Hz. The average impedanceat 1 kHz was 2.22 MX (Fig. 1a). The average phaseangle ranged from �42.65� at 100 kHz to �76.26� at10 Hz. Impedance magnitudes and phase values wereas expected for high-impedance iridium microelec-trodes in buffered saline, with impedance magnitudeillustrating the contributions of resistive circuit ele-ments both intrinsic to the electrodes and within themedium, and phase illustrating the contribution ofcapacitors and inductors.

FRAMPTON et al.1034

Impedance can also be plotted as a Nyquist spec-trum giving the relative contributions of resistance andreactance (�(frequency�1 capacitance�1)) (Fig. 1b).Bare electrodes displayed Nyquist spectra characteris-tic for iridium microelectrodes in buffered saline, withlinear slopes between 10 kHz and 10 Hz, when plot-ted on logarithmic scales. Resistance and reactance

changed in response to frequency, and reactance wasnegative due to intrinsic electrode capacitance.

Based on this standard impedance wave form andmagnitude, we were able to exclude nonfunctionalelectrodes from our analysis. Electrodes were excludedfrom future analysis if impedance displayed disconti-nuities in phase or impedance magnitudes that greatly

FIGURE 1. Characterization of baseline impedance for bare iridium microelectrodes. Impedance was measured from a total of 263iridium microelectrodes (177 lm2 per electrode, 16 electrodes per device) between 100 kHz and 10 Hz, prior to their use with tissueconstructs. Impedance data can be displayed as either Bode or Nyquist plots. (a) Bode plots displayed average impedancemagnitudes ranging from 61.484 kX at 100 kHz to 150.091 MX at 10 Hz. The average impedance at 1 kHz was 2.224 MX. Theaverage phase angle ranged from 242.65� at 100 kHz to 276.2578� at 10 Hz. Average phase angle at 1 kHz was 269.8594�.(b) Nyquist plots display impedance data in terms of reactance (y-axis) and resistance (x-axis). Nyquist profiles displayed linearslopes and values consistent with the values found for of iridium microelectrodes in buffered saline. In (a), triangles representphase angles, and circles represent magnitudes of impedance. In (a) and (b) black symbols represent the geometric means, blackbars represent standard error, and gray bars represent standard deviation.

Effect of Glial Cells on Neural Prosthetic Impedance 1035

exceeded the ranges described for bare electrodes. Inorder to reduce the number of probes used per exper-iment, electrodes could be reused after immunohisto-chemical processing and appropriate cleaning. Noappreciable changes in impedance wave form or mag-nitude were observed following our cleaning proce-dure. However, electrode failure sometimes resultedfrom mechanical stresses that occurred during theprocesses of mounting for microscopy and cleaning.Such damage included broken iridium microwires,insulation layer failure, or damage to the wire bondingor PCB. All damaged electrodes were excluded fromfuture analysis. Average bare electrode impedance didnot change over the course of our experiments, andindividual impedance measurements were consistentbetween readings.

Establishing and Characterizing Astrocyteand Microglial Cultures Surrounding Neural Probes

Cultures of either astrocytes or microglia wereestablished at a density of 100,000 cells/lL surround-ing microfabricated neural probes. For pure cultures ofastrocytes or of microglia, mean impedance magnitudeand phase values changed from the mean bare elec-trode values over the length of time in culture (Fig. 2).Differences in the time course of impedance change, asvisualized in Bode spectra for magnitude and phaseangle, existed between microglia and astrocytes(Figs. 2a–2d). Astrocytes showed an increase from thebare electrode in mean impedance magnitude at 3 daysand a further increase at 7 days in culture, which wasmaintained at 14 days (Fig. 2a). Microglia showed asimilar increase from the bare electrode in meanimpedance magnitude at 3 days. Impedance magnitudefor microglial cultures did not change between 3 and7 days in culture, but increased between 7 and 14 daysin culture (Fig. 2b). Phase angle also increased fromthe bare electrode over the course of time in culturefor astrocytes and for microglia. Astrocyte culturesinduced changes from the bare electrode in phase angleat 3 days in culture. Phase angle was increased furtherfrom the bare electrode between 3 and 7 days, and didnot increase again between 7 and 14 days (Fig. 2c).Microglia showed a larger increase from the bareelectrode in phase angle at 3 days than did astrocytes.No further changes in phase were observed at 7 or14 days in cultures of microglia (Fig. 2d).

Phase angle changes for both astrocytes andmicroglia were most apparent at lower frequencies,while changes in impedance magnitude were present atboth low and high frequency ranges. For culturesyielding phase changes that were particularly large, themagnitude of impedance appeared to change less fromthe bare electrodes at lower frequencies than at higher

frequencies. The absence of changes in the impedancemagnitude coincided with large increases in phaseangle. The changes in impedance magnitude and phaseover time in culture also appeared to follow a cell type-specific pattern, with astrocytes showing increases inimpedance magnitude and phase from the bare elec-trode at 3 days and further increases at 7 days.Microglia, however, showed large changes from thebare electrode in phase angle measured at 3, 7 and14 days, and relatively small changes from the bareelectrode in impedance magnitude at early time points.

After 14 days in culture, the final impedance mea-surement time point, cultures were fixed and processedfor immunohistochemistry and microscopy. Analysisof the alginate tissue constructs surrounding thedevices showed dense populations of process-bearingcells, for both the microglial and the astrocyte cultures.The absence of microglial labeling in astrocyte cul-tures, and astrocyte labeling in microglial cultures,indicated that we were able to produce relatively purecultures of each glial cell subtype. Astrocytes sur-rounding the device appeared to be in a reactive state,based on GFAP expression and occurrence of cellmorphologies that were dissimilar to the stellate mor-phologies observed in normal brain tissue. However,astrocytes in culture have been shown previously tobe constitutively reactive by labeling with GFAP(Fig. 2e). Microglia displayed either ramified orameboid morphology and formed dense networkssurrounding the devices (Fig. 2f). Morphologicalcharacteristics, such as the presence of processes in themajority of cells, indicated that the culture systemremained viable for the 14-day period over which timeimpedance measurements were recorded.

Generation of Mixed Glial Cultures

In order to produce sufficient numbers of microglia,up to four, 100 mm2 culture dishes of cells wererequired. This provided enough cells to produce thedesired seeding ratios at cell densities similar to that ofbrain tissue. We were able to produce defined ratiosby pre-counting of glial cells and subsequent centri-fugation to generate a pellet for use in alginate tissueconstructs. Ratios of astrocytes to microglia werechosen based upon the cellular ratio in rat cortex:approximately 75% astrocytes to 25% microglia.7 Inorder to assess the effects of cell ratio on glial cellreactivity and tissue impedance, we cultured microgliaat not only 25%, but also at 12.5 and 50%. The dif-ferent ratios were tested in order to more effectivelyevaluate the contributions of each cell type to mea-sured impedance. The relative proportions of astro-cytes to microglia could be readily gaugedsurrounding probes at 3 days (Figs. 3a–3c), a time

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FIGURE 2. Pure cultures of astrocytes and microglia affect the electrode impedance measured over 14 days. Impedance mea-surements were recorded at 3, 7 and 14 days from high-density cultures composed entirely of either astrocytes or microgliasurrounding neural probes. (a–d) Bode plots display changes in the impedance modulus over time in culture, for both astrocytes(a, b) and microglia (c, d). (a) Astrocyte cultures reached their maximum impedance at 7 days, while impedance modulus formicroglia (c) increased only slightly at 3 and 7 days relative to the bare electrode, and continued to increase relative to the bareelectrode at 14 days. (b, d) Bode plots of phase angle show differences in phase angle over time and between cultures. Microgliashowed larger changes in phase angle relative to the bare electrode than did astrocytes. Circles represent impedance modulus andtriangles represent impedance phase. Color designates time point in culture. Bars denote SEM. (e, f) Cell morphology and dis-tribution in the vicinity of electrode sites could be observed by immunohistochemical labeling and microscopic detection of GFAP(red) (e) and iba-1 (green) (f). Scale bar 5 50 lm.

Effect of Glial Cells on Neural Prosthetic Impedance 1037

point before significant cell proliferation. It is evidentfrom representative images that the desired cell den-sities and proportions of astrocytes and microgliawere achieved.

Cellular Distribution Surrounding Electrodes

Astrocytes and microglia showed differences in theircharacteristic distributions within 3D tissue constructs.Microglia were observed either as single cells, or asgroups surrounding clusters of astrocytes. Microgliawere also located closer to the probe surface in cultures(Figs. 3a–3c). Astrocytes often formed clusters of cellsthat were located in close proximity to the electrodesites as well as at distances further away from theprobes (Fig. 3a). Both cell types were observed toattach directly to the surface of the probe and on-boardelectrodes. The distributions of Iba-1-labeled microgliawere consistent with the distributions of observedED1-labeled reactive microglia/macrophages surround-ing microfabricated probes implanted in rats.4 In ourin vitro culture system, the attachment of cells directlyto the probe surface may have been partially caused bycellular adhesion to the positively charged lysineresidues used to promote attachment of the alginatematrix, and also by cellular reaction to the rigid siliconsubstrate within the alginate. Although some cellsattached to the surface of the probe, most cells inculture were located within the 3D alginate matrixsurrounding the device. Tissue constructs containingastrocytes and microglia covered the entire length ofthe neural probes (Figs. 3a–3c).

Impedance Characterization of Mixed Glial Cultures

Impedance measurements were made using variousratios of glial cells at 3, 7, and 14 days, to determinethe effect of cell type on impedance. Data were plottedas impedance magnitude or phase vs. frequency (Bodeplots) to enable differences in impedance, between thebare electrode values and the values for the various cellseeding conditions to be visualized (Fig. 4). Differencesfrom the bare electrode impedance spectra wereobserved at 3 days for all cell seeding conditions,consistent with previous findings that derived fromhigh-density cultures of glioma cells (Figs. 4a and 4b).Impedance magnitude increased from the bare elec-trode, in the frequency range between 10 kHz and100 Hz for all cell conditions (Fig. 4a). Phase anglealso exceeded the bare electrode angle for all three cellseeding conditions (Fig. 4b). At 3, 7, and 14 days,cultures rich in astrocytes (12.5 and 25% microgliacultures), showed larger increases in impedance mag-nitude from the bare electrode than did 50% microgliacultures; however, all cell seeding conditions showed

FIGURE 3. Mixed cultures of astrocytes and microglia wereused to model neural tissue impedance in vitro. Glial cellswere cultured surrounding neural probes, at various ratios ofastrocytes to microglia, to permit interactions between glialcell types and provide physiologically relevant culture con-ditions. 3D image sets were collected from whole mounts ofconstructs and probes, labeled for GFAP (red), Iba-1 (green)and TOTO-3 (blue). Confocal images confirmed that cell cul-tures contained the desired densities and ratios of cells in 3D.For all seeding conditions, microglia were located close to thetissue construct-device interface. Both astrocytes andmicroglia were observed to attach and extend processeswithin the matrix, as well as directly along the probe andelectrodes. (a) 12.5% microglia, 87.5% astrocytes at probe tip.(b) 25% microglia, 75% astrocytes at probe mid-shank. (c) 50%microglia, 50% astrocytes at probe shank proximal to PCB. Allx–y and x–z images are sets of maximum intensity projectionsof confocal image volumes. The white trace indicates thelocation of the probe and electrodes (superimposed outlinewith circles). Scale bar 5 50 lm.

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FIGURE 4. Bode plots of impedance measured from electrodes surrounded by mixed cultures of astrocytes and microglia displayspectral differences among three cell seeding conditions. Impedance modulus and phase were recorded for each of the 3 mixed-glia seeding conditions at 3, 7 and 14 days in culture, and were plotted vs. frequency. (a) At 3 days, cultures rich in astrocytes (12.5and 25% microglia) showed the largest increase in impedance modulus compared to the bare electrode impedance. (b) At 3 days,all seeding conditions showed similar increases in phase angle, compared to the bare electrode phase angle. (c, d) Increases inimpedance modulus (c) were maintained for all cell seeding conditions at 7 days. Differences in phase angle (d) between cellseeding conditions were observed. (e) At 14 days, cultures of 25% astrocytes showed the largest increase in impedance moduluscompared to the bare electrode. (f) Clear differences in phase between seeding conditions were observed at 14 days, with culturesof 50% microglia showing the largest increases from the bare electrode at lower frequencies. For all plots circles representimpedance modulus and triangles represent phase angle. Color designates cell seeding condition. Bars denote SEM.

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similar increases in phase angle from the bare electrodephase angle.

At 7 and 14 days in culture, differences in imped-ance magnitude from the bare electrode value weremaintained for all seeding conditions, with the 25%microglia condition showing the largest gain inimpedance magnitude (Figs. 4c and 4e). At 7 and14 days impedance magnitude showed smaller differ-ences from the bare electrode values at low frequencies.This was associated with changes in the phase angle,and occurred in the same frequency range as wasobserved for pure cultures of astrocytes and microglia.At 7 and 14 days in culture, clear differences wereobserved in phase angle among the three seedingconditions (Figs. 4d and 4f). At 14 days, cultures of50% microglia showed the largest overall increases inphase angle (Fig. 4f). For all cell seeding conditions,impedance and phase angle either increased or weremaintained through 14 days in culture.

These differences in the impedance spectra can bereadily observed as Bode diagrams for impedancemagnitude or phase. For comparison with results fromother studies on neural tissue impedance, and to pro-vide an additional method for visualizing relativechanges in resistance and capacitance (as reactance) weplotted impedance data as Nyquist spectra (Fig. 5) forimpedance data recorded at 3, 7, and 14 days in culture.As was the case for the Bode plots, the Nyquist plotsshowed clear differences in impedance between each ofthe cell seeding conditions and the bare electrode at3 days (Fig. 5a). Differences between cell seeding con-ditions were more apparent at 7 and 14 days in culture,with the 50% microglia cultures showing the largestshift in reactance at 14 days (Figs. 5b and 5c). Changesin the impedance were consistent with the findings ofprevious reports, and showed changes in reactance(y-axis shifts) indicating changes in capacitance andincreases in resistance (x-axis shifts). These results,together with the results from Fig. 2, indicate that bothastrocytes and microglia contribute to changes in theimpedance measured from neural probes at differenttime points in culture.

Visualization of 3D Cultures of Mixed Astrocytesand Microglia After Impedance Measurements

Cultures were fixed immediately after the finalimpedance measurements at 3, 7, or 14 days in culture.Immunohistochemical and microscopic analysisrevealed changes in the tissue constructs over time. At3 days (Fig. 6a), glial cells were distributed as singlecells or in small clusters. Both astrocytes and microgliadisplayed rounded morphology with outgrowth offine processes. At 7 days in culture (Fig. 6b), astro-cyte (green) clusters increased in size, and larger

proportions of both the astrocytes and the microgliadisplayed outgrowth of processes. At 14 days (Fig. 6c),many cultures exhibited robust process outgrowthfrom astrocytes. Glial cells were observed to formcontiguous networks of cells surrounding the deviceand the electrodes. Throughout the 14-day cultureperiod, almost all microglia remained ameboid inmorphology, indicative of a persistent state of reac-tivity within the tissue construct surrounding thedevice electrodes. Astrocytes, often found in clusters,displayed extensive process outgrowth over time inculture. Increases in cluster size may have resultedfrom proliferation of astrocytes.

Analysis of Impedance Changes

Bode and Nyquist plots display bare electrodeimpedance averaged for all electrodes at each timepoint, but may obscure the relative changes betweenthe bare electrode values and the values for the variouscell seeding conditions. Shaded matrix plots allowedthe visualization of relative changes in the impedanceamong test conditions. Matrix plots presented nor-malized impedance data at defined frequencies as afunction of cell seeding ratio and time in culture(Figs. 6–8). Four frequencies (10 kHz, 1 kHz, 100 Hz,and 10 Hz) were selected for further analysis to enableobservation of changes in impedance over multiplespectral regions.

As frequency decreased, relative changes in imped-ance magnitude (Fig. 7a) generally decreased, andrelative changes in phase angle (Fig. 7b) generallyincreased. Over time in culture, impedance magnitudeincreased relative to the magnitude for the bare elec-trode, peaking at 7 and 14 days for most conditions.Pure cultures of either astrocytes or microglia andcultures of 25% microglia showed the largest increasesover time. All culture conditions with the exception ofpure cultures of microglia showed at least a twofoldincrease in relative impedance magnitude over theentire frequency range. Phase angle changes relative tothe angles for the bare electrodes were more prominentat low frequencies and did not show a dependence ontime in culture. Conditions seeded with high propor-tions of microglia (50% and pure microglia cultures),showed larger increases in phase angle at lowfrequencies.

In order to assess statistical differences in impedancemagnitude and phase as a function of glial cell seedingratio, EIS data were fitted using non-linear leastsquares regression. The Y0 curve fitting outputparameters were used to compare statistical differencesbetween conditions. At 3 days there were no significantdifferences between cell seeding conditions for eithermagnitude or phase, although all values were significantly

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FIGURE 5. Nyquist spectra allow changes in the reactance and resistance to be described from impedance data recorded atvarious cell seeding conditions. Impedance data were compiled and plotted as Nyquist spectra of resistance vs. reactance.Changes in the resistance and reactance were consistent with Bode plots of impedance, as well as with previous in vitro andin vivo observations. Nyquist spectra are shown for cultures of mixed glia at (a) 3 days, (b) 7 days and (c) 14 days. Color designatescell seeding condition. Bars denote SEM.

Effect of Glial Cells on Neural Prosthetic Impedance 1041

FIGURE 7. Bode impedance plots were normalized to theimpedance recorded from the bare electrodes, for determi-nation of relative changes in impedance among test condi-tions. Shaded data matrices can be used to identify changesin the relative impedance compared to bare electrodes. Bodeimpedance plots were normalized to the impedance recordedfrom the bare electrodes, for determination of relative chan-ges in impedance among test conditions. Four frequencies(10 kHz, 1 kHz, 100 Hz, and 10 Hz) were selected for furtheranalysis. Electrode impedance values were normalized bysubtracting bare electrode impedance from test impedanceand dividing the result by the bare electrode impedance forindividual electrodes. Relative changes were plotted as meanvalues, to facilitate recognition of trends across cell types,frequencies, and time points. (a) Pure cultures of eitherastrocytes or microglia, and cultures of 25% microglia,showed the largest relative increases in impedance modulusover time, displayed as peaks in the impedance magnitudeplots. All culture conditions with the exception of pure cul-tures of microglia showed at least a twofold increase in rela-tive impedance magnitude over the entire frequency range.(b) Phase angle changes relative to the bare electrode did notshow as much of a dependence on time in culture. However,conditions rich in microglia (50% and pure microgliacultures), showed the largest increase in phase angle. Colorsrepresent the relative impedance change from the bareelectrodes.

FIGURE 6. 3D cultures of mixed glial cell types form sheathsaround neural probes over 2 weeks in culture. Under all cul-ture conditions tested, astrocytes and microglia extendedprocesses and showed signs of cell proliferation within theconstructs over time. At 2 weeks in culture the 25% astrocyteseeding condition displayed the largest changes in magni-tude of impedance. Shown are representative images of 25%astrocyte cultures at 3 (a), 7 (b) and 14 (c) days in culture. Allx–y and x–z images are sets of maximum intensity projectionsof confocal image volumes. Red labeling corresponds toastrocytes (GFAP), green to microglia (Iba-1), and the whitetrace to the approximate location of the probe (superimposedoutline with circles).

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larger with respect to bare electrode values (Figs. 8a and8b). As a function of time in culture, several conditionsshowed sustained changes in impedance magnitude andphase with respect to the bare electrode. The 25%microglia seeding conditions maintained large changesin impedance magnitude and phase over 14 days inculture, and the 50% microglia cell seeding conditionmaintained significant changes in phase angle (Figs. 8cand 8e).

These results lead to an interpretation that celldensity is a more critical factor than is cell type indetermining the extent of impedance change arounddevice electrodes. However, the effects of cell type onimpedance are dynamic over 14 days in culture. TheEIS and immunohistochemical data are consistent witha scenario in which at early time points (3 days)impedance changes are mediated by microglia, the celltype observed closest to electrodes. At later time pointsastrocytes may contribute to sustained changes inimpedance through proliferation and further occlusionof extracellular space.

DISCUSSION

In this report we demonstrate methods to culturevarious ratios of astrocytes and microglia in 3D con-structs at high density, thus producing test systems thatare similar in cell density to that observed in vivo in ratcortex. Moreover, based on observed changes in cellmorphologies and distribution, as well as previous live/dead and mitochondrial activity analysis,15,16 wereport high cell viability and function throughout theduration of our experiments (2 weeks). By buildingthese constructs around microfabricated neuralprobes, and through the use of tissue impedancespectroscopy, this culture system permits measurementof resistance and capacitance through tissue constructsranging in thickness from 50 to 100 lm. Thus this 3Dsystem provides a model of brain tissue aroundinserted neural probes and has enabled us to test ourhypothesis that individual cell types make distinctcontributions to the observed changes in complextissue impedance spectra. Previous experiments deter-mined that density of cells surrounding the neuralprobe can by itself affect the impedance measured fromelectrodes.16 The present data support our cell-typecontribution hypothesis through the observed time-dependent changes in cell organization and dynamicimpedance changes.

Tissue Constructs and Cell Distribution

The high cell density chosen for use in theseand previous experiments is similar to the density

determined in rat cortex by quantitative mapping of avariety of neural cell types.7 Our previous study usingthe LRM55 astroglioma cell line25 required severalmedium changes per day, to remove undesirable wastebyproducts and to sustain cellular metabolic demands.The primary cell culture model used here requiresmedium changes only twice per day, due to slowerrates of cell metabolism and proliferation. This per-mitted maintenance of viable cells through 2 weeks,enabling measurement of impedance at multiple timepoints.

Functionalization of the alginate constructs bycovalent linkage of the integrin receptor ligand peptideepitope RGD, promoted cell attachment to the matrix,maintenance of 3D cultures with relatively even celldistribution, and extension of cell processes in 3D. Theoptical transparency of the alginate tissue constructspermitted microscopic visualization of cell morpholo-gies and assessment of the distribution of cells inrelation to the neural probe.

Microglia, particularly at early time points, tendedto be distributed closer to the probe-tissue constructinterfaces, while clusters of astrocytes were distributedthroughout the volume of the tissue constructs. Onepossible explanation for the differences in cell distri-bution is that microglia, often not clustered, sedimentthrough the viscous alginate prior to crosslinking.Alternatively, microglia may migrate toward the probesite after alginate crosslinking, while astrocytes do notmigrate. This could be and inherent difference in theproperties of astrocytes and microglia or could be dueto the observed organization of astrocytes into clustersthat are relatively less mobile due to their larger size.The high cell density used for our experiments maygive rise to a more open structure than other alginatemodels. While the pore size of alginate is generally toosmall to allow movement of cell bodies, it could alsobe possible that high density cultures of microgliaremodel the construct environment and move toward thedevices in our in vitro model. Microglia rapidly migratethrough tissue in response to biochemical gradientsand other environmental cues.19 Microglia have alsobeen observed to be the first layer of cells aroundinserted neural probes in vivo.4 Thus, our observationsofmicroglial redistribution in our 3D constructs provideevidence of how well this system mimics the cell distri-bution observed in vivo and in other in vitromodels.

Iba-1 was chosen as a specific marker for microglia inthis study because immunolabeling of this protein isobserved as a uniform fluorescent signal throughout theentire cell independent of the reactive state of the cells.7

However, in comparison to the work of Biran et al., theIba-1 signal observed in our in vitro system showed asimilar distribution to the ED1 labeled activatedmicroglia and macrophages in vivo5,6,36,38. The role of

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peripherally derived macrophages could be investigatedby including macrophages in future 3D modelingexperiments.

The probe itself may provide cues for the movementand attachment of cells in our culture system. It is clearthat cells attach to devices in vivo.38,39 In order to

FIGURE 8. Nonlinear least squares regression was used to fit impedance data and identify significant differences betweenconditions. Several conditions were selected for detailed statistical comparisons. (a, b) At 3 days both the impedance modulus andphase angle were significantly higher than the bare electrode values for all three cell seeding conditions, as determined from the Y0

parameters calculated during regression analysis. (c, d) Over time in culture the 25% astrocyte condition maintained the largestincreases in both impedance modulus and phase. (e) Statistically significant increases in impedance phase were also observed forthe 50% microglia condition through 14 days. In combination with immunohistochemical data, these trends suggest that activatedastrocytes contribute to sustained changes in impedance magnitude and phase, while microglia may contribute to more acutechanges as well as prolonged changes in phase angle. Error bars denote SEM. p < 0.01 was considered to be significant byStudent’s t test. Colors denote cell seeding condition as represented in Figs. 4 and 5.

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permit alginate attachment to the neural probes,devices were treated with poly-L-lysine, thus providinga positive surface charge that will provide an appro-priate surface for cell attachment. There are severalpossible mechanisms that may promote the observedmicroglia distribution including the charged probesurfaces, the mechanical differences between the rigidsilicon probes and the more elastic alginate matrix,preferential cell attachment to the rigid silicon sub-strate, or cell-mediated response promoted by thepresence of a foreign body, e.g. probe.

Changes in Tissue Construct Impedance

Complex impedance spectroscopy is an effectivetechnique for evaluating the electrical properties ofdevice–tissue interfaces.16,26,41 Using our 3D tissueconstructs, we are able to compare changes in imped-ance spectra with changes in artificial tissue constructswith different types of cells, microglia vs. astrocytes,and different ratios of microglia and astrocytes. Thuswe inferred the contributions made by each cell type tothe resistive and capacitive changes. Such informationwill provide a method for real-time on-line diagnosis ofdevice–tissue interactions. Time points of 3, 7, and14 days were chosen for analysis, to allow comparisonwith the results of previous experiments, and to mini-mize the damage to cells that arises from repeatedremoval from the incubator and impedance testing.Impedance changes were found to be dependent uponseveral factors, including test-pulse frequency, time inculture, and cell seeding condition.

Impedance changes occurred over time in culture.For all seeding conditions, impedance magnitude andphase angle increased relative to the bare electrode.These changes over time may have resulted from thecontinued growth of cells within the alginate matrixincluding process outgrowth, attachment to devices,proliferation, and production of extracellular matrixmaterials. Results from confocal imaging provide cleardemonstration that the first factors contribute to theobserved impedance changes. We did not performexperiments to quantify proliferation or test forextracellular matrix production.

Cell type also had an effect on impedance measuredfrom devices. The largest differences were observed forpure populations of either microglia or astrocytes. Itwas evident, from inspection of confocal images takenof tissue–device constructs, that pure cultures formedmore contiguous cell networks around devices, whilemixed cultures demonstrated more complicated cellorganization. The organization of mixed cultures mayresult from cell–cell interactions and cell responses toneural probes. It is also possible that cell behavior in

the mixed cultures resulted from additional experi-mental variables, especially increased numbers of stepsin construct preparation.

Increases in impedance magnitude relative to bareelectrodes were observed for all mixed cultures; thelargest changes occurring with the 25% microgliaseeding condition. This seeding condition may mostclosely approximate cell proportions observed in vivo,and thereby provides an environment that best pro-motes interactions between astrocyte and microglia,resulting in enhanced glial reactivity. Cultures rich inmicroglia (50% and pure) showed the largest changesin phase angle from the phase angle of the bare elec-trode, occurring at 3 days and persisting through2 weeks in culture. These findings support an inter-pretation that microglia recognize and surround neuralprobes at early time points, and then attach directly toelectrodes, causing changes in electrode impedance. Inthis scenario astrocytes contribute more to changes inelectrical impedance by extending processes and pro-ducing extracellular matrix in the tissue surroundingdevices, eventually resulting in the formation of aninsulating sheath. This interpretation is consistent withboth our impedance data and the observed changes inthe cell distributions.

Comparison to Other In Vitro and In VivoMeasurements

The impedance measurements described in thisreport verify and expand upon previously reportedin vitro impedance evaluations. In a previouslydescribed 2D culture system, the effects of a variety ofcell types cultured as monolayers on the impedance ofiridium electrodes was evaluated.26 Impedanceincreases of between 20 and 80% of the bare electrodevalues were described. In this report we focused onfewer cell types, namely astrocytes and microglia, andevaluated their effects on electrode impedance whencultured in a 3D environment at tissue-like densities. Inour constructs it was possible for microglia andastrocytes to interact in a 3D environment and to formdifferent distributions as a function of time in culturethroughout this volume. In accordance with Merrilland Tresco, impedance was observed to increaseat least more than 50% from the values for bare elec-trodes for most cell seeding conditions.26 For some cellseeding conditions these changes were maintained overthe course of 14 days.

The impedance changes that we observed can becompared to previous in vitro findings, as well as tomeasurements made in vivo.41 Impedance changes areoften characterized by shifts in the phase angle andmagnitude of impedance, apparent when the data aredisplayed as either Bode or Nyquist spectra. At 3 days

Effect of Glial Cells on Neural Prosthetic Impedance 1045

in culture increases in impedance magnitude relativeto the bare electrode were similar in extent to theimpedance changes observed in previous experimentsinvolving 3D cultures of astroglioma cells; and weremaintained at 7 and 14 days in culture. Although theextent of impedance change relative to bare electrodeswas less than observed in vivo, similarities in the fre-quency-dependent spectral profiles were apparentwhen Nyquist spectra were compared.

Discrepancies between changes observed in vitroand those observed in vivo may result from severalfactors including the volume of tissue assessed, theamount of resistive matrix present, and the presence ofadditional cell types (e.g. neurons, oligodendrocytes/myelin, macrophages and endothelial cells/blood ves-sels). These cell types are not part of the current in vitromodel. Neuronal fibers as well as blood vessels mayeither act as low resistance pathways for current todissipate, or may provide additional resistance andcapacitance caused by tight junction interactionsbetween endothelial cells or the insulation properties ofmyelin. Another difference between our model andin vivo experiments is that our model does not involvedevice implantation, thus we have eliminated initial celland tissue damage resulting from this perturbation.This test system may more closely mimic the reactiveresponses that occur due to the sustained presence of adevice rather than the responses associated with deviceimplantation, cell damage, compromised blood–brainbarrier and effects of soluble factors and reactiveoxygen species.

Differences in probe design, including variations inprobe materials and dimensions, have also been shownto cause variability in tissue reactivity.5,6,36 Addition-ally, electrode material has an effect on the initialimpedance of the electrodes and these differencescomplicate comparison of impedance spectra.17 Previ-ous experiments in vivo were carried out using largetungsten wire electrodes that displayed low initialimpedances.41 The electrodes used in our in vitroexperiments were microfabricated using iridium andhad large initial impedances, limiting the volume oftissue that could be assessed, and possibly maskingimpedance changes caused by cells.

Finally, in the present study it is not possible todetermine whether or not the impedance changeswould affect acquisition of neuronal recordings.Therefore, future in vitro applications should not onlyinclude glial cells but also neurons to directly assess theability of the electrodes to form stable recording ofneuronal electrical activity. Additional analysis ofimaging data could also provide more complete mapsof cell distributions and thus provide data that wouldenable evaluation of more complete mathematicalequivalent circuit models needed to deduce the resistive

and capacitive elements from impedance spectra. Suchmethods will permit more accurate comparison ofresults between in vitro and in vivo preparations.Additional studies could also be designed to studyinsertion of devices into 3D matrices to study theresponse to neural damage as well as to monitor cellmovement by live imaging.

CONCLUDING REMARKS

We have presented a 3D in vitro model capable ofnot only assessing the effects of cell densities, but alsothe dynamic effects of primary cell types on electrodeimpedance. The ability to model tissue for measure-ment of impedance in vitro provides a valuable tool fordeconstructing the cellular and tissue mechanismsunderlying tissue reactivity to implanted neural probes,as well as assessing the performance of neural pros-thetic devices in the central nervous system. The resultsfrom these experiments suggest that this model will bea valuable tool for future assessment of neural pros-thetic device performance.

ELECTRONIC SUPPLEMENTARY MATERIAL

The online version of this article (doi:10.1007/s10439-010-9911-y) contains supplementary material,which is available to authorized users.

ACKNOWLEDGMENTS

The authors would like to acknowledge theWadsworth Center Advanced Light Microscopy andImage Analysis Core and its director, Richard Cole.This work was supported in part by the Nanobio-technology Center (NBTC) an STC program of theNSF under agreement number ECS-9876771, byNIBIB (R21R21EB007782), and by NIH (NS R01-04488145).

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