Exploiting advances in imaging technology to study biofilms by applying multiphoton laser scanning...

Journal of Microscopy, Vol. 235, Pt 2 2009, pp. 128–137

Received 9 February 2009; accepted 20 April 2009

Exploiting advances in imaging technology to study biofilms byapplying multiphoton laser scanning microscopy as an imagingand manipulation tool

M . A . L A K I N S ∗, J . L . M A R R I S O N†, P . J . O ’ T O O L E†& M . W . V A N D E R W O U D E ∗∗Centre of Immunology and Infection, Department of Biology, University of York and The Hull YorkMedical School, PO Box 373, York YO10 5YW, U.K.

†Imaging and Cytometry Laboratory, Technology Facility, Department of Biology, University ofYork, PO Box 373, York YO10 5YW, U.K.

Key words. Biofilm, Escherichia coli, imaging, method, multiphoton laserscanning microscopy.

Summary

Biofilms are an important element of the natural ecosystemsbut can be detrimental in health care and industrial settings.To improve our ability to combat biofilms, we need tounderstand the processes that facilitate their formation anddispersal. One approach that has proven to be invaluableis to image biofilms as they grow. Here we describe toolsand protocols to visualize biofilms with multiphoton laserscanning microscopy, compare this with single photon laserscanning confocal microscopy and highlight best workingprocedures. Furthermore, we describe how with multiphotonlaser scanning microscopy the laser can be used to manipulatethe biofilm, specifically to achieve localized bleaching, killingor ablation within the biofilm biomass. These applicationsopen novel ways to study the dynamics of biofilm formation,regeneration and dispersal.

Introduction

A biofilm consists of a community of cells that is attached to asurface and enclosed within an extracellular matrix. Biofilmscan be beneficial or detrimental, for example enhancing wastewater treatment or by causing pipe fouling, respectively. Inaddition there is significant interest from the health care fieldwhere biofilms lead to persistent and difficult to eradicateinfections. The importance of biofilms in these and relatedareas have led to concerted efforts to understand what a biofilmis, how it is formed and what its properties are.

A generic working model for biofilm formation has beendeveloped over the past decade (O’Toole et al., 2000; Ghigo,

Correspondence to: Marjan van der Woude. Tel: 44-(0)1904-328841; fax: 44-

(0)1904 328844; e-mail: [email protected]

2003; Beloin & Ghigo, 2005). Up until now biofilm imaginghas concentrated on identifying and understanding thesequence of events leading to biofilm formation and it’sstructure (Stoodley et al., 2001). Imaging biofilms has alsohelped us understand how a biofilm can protect a subset ofthe cells from the activity of antimicrobial agents (Donlan &Costerton, 2002; LaFleur et al., 2006; Anderson & O’Toole,2008; Folkesson et al., 2008). An important feature for manyproperties of a biofilm is that it consists of a heterogeneouscommunity and imaging is an invaluable tool to identifyand understand this aspect. Imaging has also helped identifyhow variables, both biological and abiotic (Ghigo, 2001;Horswill et al., 2007; Goller & Romeo, 2008) alter biofilmformation and its properties. Finally, imaging has facilitatedthe study of the dynamics and evolutionary processes betweenspecies in a biofilm (An et al., 2006; Hansen et al., 2007). Asignificant part of this understanding has been obtained fromstudies with single photon laser scanning confocal microscopy(SP-LSCM). For example, the understanding of biofilmformation by Pseudomonas aeruginosa has been enhanced bydetailed imaging studies (Heydorn et al., 2002; Klausen et al.,2003a). Yet, all of these events and processes are far frombeing completely understood.

Multiphoton laser scanning microscopy (MP-LSM) can bea valuable additional tool to study biofilms. To date, thisspecific imaging technology has been used for the analysis ofbacterial biofilms in only a limited number of studies (Vroomet al., 1999; Bryers, 2001; Neu et al., 2002; Hu et al., 2005;Lacroix-Gueu et al., 2005; Briandet et al., 2008). Yet MP-LSM offers well-known advantages and benefits that can alsolead to further advances in microbiology as was reviewed inthe context of microbial ecology (Neu et al., 2004). Theseadvantages include deep imaging of biological material due

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to the nature of excitation of fluorochromes and confinementof the excitation to a defined focal volume. Compared to SP-LSCM, MP-LSM with non-descanned detectors (NDD) has theadvantage that the longer wavelength used for excitationpermits deeper penetration and inflicts less biological damagewhen used correctly, and furthers applications in live tissues(Yuste, 2005; Mansson et al., 2007).

An additional benefit of MP-LSM is that the underlyingtechnology allows new approaches to be developed that utilizethe laser as a manipulation tool. Some of these approaches,such as photobleaching experiments, are well established ineukaryotic and even microbial cell biology (Carballido-Lopez& Errington, 2003; O’Toole, 2003; Yuste, 2005; Giepmanset al., 2006; Barak et al., 2008). However, such approacheshave rarely been applied to biofilms (Bryers & Drummond,1998; Neu et al., 2004) and certainly not in conjunction withother potential applications and benefits of MP-LSM. In orderto maximize the benefits of MP-LSM for biofilm imaging, and todevelop novel approaches to manipulate the biofilm material,a more complete appreciation and understanding of both thecomplexity of the technology and the biological material isrequired.

To enhance and optimize the use of the advanced MP-LSM imaging technology in the field of biofilm research, weprovide and rationalize the use of protocols with an emphasison gathering high-resolution data in model biofilms. Typicaltechnical and biological factors affecting optimal applicationof MP-LSM, i.e. image quality and image interpretation, arediscussed in the context of bacterial biofilm analysis. Someimage artefacts may be much more relevant and more difficultto identify for bacterial analysis as compared to eukaryotic cellbiology due to the nature of the material. We have comparedSP-LSCM versus MP-LSM with different detector systems anddescribe how localized bleaching, killing and ablation ofregions of interest in a biofilm can be achieved. We discusshow this may lead to novel approaches to address questionsin the field. It is hoped that this will facilitate use of thistechnique among microbiologists and generate appreciationof the biofilm-specific biological variables for imaging experts,and thereby initiate advances in the field of biofilm research.

Materials and methods

Bacterial strains, media and culture conditions for biofilms

All biofilms consisted of derivatives of strain Escherichia coli (E.coli) TG1 18, which constitutively produces the F pilus (Ghigo,2001). This strain was transformed with plasmids pZEtetR21-GFP (Green Fluorescent Protein) (Da Re et al., 2007) orpRSETB-tdTomato (Shaner et al., 2004) resulting in strainsMV1257 and MV1264, respectively. The strains were grownin M9 minimal medium with 0.4% glucose at 37◦C (Ghigo,2001; Reisner et al., 2003). A total of 30 μg mL–1 kanamycinand 100 μg mL–1 ampicillin were added to maintain the

plasmids pZE21-GFP or pRSETB-tdTomato respectively, butwere omitted from the medium during biofilm growth. Therewas no evidence of plasmid loss.

In the flow cell, a biofilm up to 180 μm thick was obtainedreproducibly within 48 h after seeding with approximately1×108 log-phase cells. Gravity facilitated adhesion to thecover slip occurred over 30–60 min, which was followed by‘flushing’ of unattached cells for 5 min using a high flow rate ofapproximately 10 mL min–1. The flow rate was then adjustedto 1 mL min–1 using a Watson–Marlow 205 U pump. To obtainhigh-quality images, the flow cells (see below) were assembledwith #1.5 cover slips. A bubble trap was placed between thepump and the flow cell. Growth medium and flow cell werekept at 37◦C at all times. We also imaged biofilms grownunder static conditions in 35-mm Glass Bottom Culture Dishes(MatTek Corporation, Ashland, MA, U.S.A.).

Stains

Fluorescence from GFP- or tdTomato-expressing strains wasanalysed as indicated above. Alternatively, the BACLightLIVE/DEAD stain (Invitrogen, Paisley, U.K.) (1 mL of 1:1000in media) was added just prior to imaging. This stain consistsof SYTO9 (green) that is taken up by all cells, and propidiumiodide (PI) (red) that is taken up by cells with a compromisedmembrane. With the strain and growth conditions usedhere the fluorochromes completely penetrated the biofilmbiomass.

The flow cell and bespoke hardware

A reusable, autoclavable flow cell with dual growth chambers(47.5 mm×12.7 mm×1.6 mm) fitted with injection ports wasused (MODEL FC 281; Biosurface Technologies, Bozeman, MT,U.S.A.). The microscope stage-mounting tab was removed andmodified with a widened viewing window to facilitate accessof the Plan-Apochromat 63×/1.4 NA oil DIC (DifferentialInterference Contrast) objective and other high NA lenses.Because this is an adaptation of a commercially availableproduct by the same company this hardware is accessible toall research groups. Additional bespoke hardware includeda mount to accommodate the assembled flow cell with portsand tubing on the microscope stage, and two custom madeobjective risers (University of York, Department of Biologyworkshop). Technical details of the custom-made hardwareare available upon request.

Microscopy

Imaging was carried out on a Zeiss LSM 510 NLO MetaLSCM using a bespoke configuration with three NDD andcoupled to a Coherent Chameleon Ultra laser (Coherent, Inc.,Santa Clara, CA, U.S.A.). This was fitted onto a Zeiss Axiovert200 microscope (Carl Zeiss Ltd, Welwyn Garden City, U.K.) in

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a fully enclosed, blacked out, temperature controlled chamber(Solent Scientific, Segensworth, U.K.).

Images were acquired using various objectives including aPlan Apochromat 63×/1.4 oil DIC objective, C-Apochromat40×/1.1 W Corr UV-VIS-IR objective, C-Achroplan NIR40×/0.8 objective and a W Plan Apochromat 20×/1.0 DICVIS-IR objective. Excitation wavelengths were assessed andall multiphoton experiments were performed at 800 nmunless otherwise stated. For all non-descanned multiphotonexperiments a HFT KP 650 short pass primary dichroicwas used and emission wavelengths shorter than 490 nmwere reflected into NDD1 with a FT490 dichroic anddetected through a 435–485 nm band pass emission filter.Wavelengths longer than 490 nm were transmitted throughthe FT490 dichroic and split by a FT560 dichroic beforedetection with a 575–640 nm band pass emission filterin NDD2 and a 500–550 nm band pass emission filter inNDD3. For multiphoton and single photon experiments usingthe internal scan head detectors a HFT KP 650 or a HFT488/561 primary dichroic was used, respectively, along witha NFT565 secondary dichroic filter. GFP and SYTO9 emissionwas detected through a 500–550 nm band pass emission filterand PI emission was detected through a 575 nm long passemission filter. Image analysis was carried out using Volocity(Improvision) software. Image grey levels were optimized forpublication in Adobe Photoshop with all images in a figuretreated the same.

The post-lens laser power applied for each image obtainedwas determined using a Fieldmate meter and a PM2sensor (Coherent). Laser output was calibrated post-lens forcomparison studies and used as a guide for laser powers usedin the various experiment types.

In prolonged time-lapse experiments 50 μm z-stacks (z-slices taken at 1 μm intervals) were acquired automaticallyevery 30 min during two consecutive 24 hour periods.However, during these long imaging periods and at the highmagnification used the pulsed flow from the pump was visibleas minor perturbations in the image. To overcome this eachfield in the z-stack was imaged twice and frame averagingapplied. This allowed the pump to remain on during extendedimaging periods preventing any associated biological sideeffects and avoiding the practical problems of coordinatingthe automated imaging and pump control.

Laser-mediated manipulations

Selective bleaching and killing of the cells was induced byonly scanning a defined region of interest (ROI) with avarying number of iterations at a slightly elevated laser powercompared to standard imaging conditions (typically 27 mWfor normal imaging and 125 mW for killing). For ablation, asimilar ROI was used and the laser power was increased to175 mW to induce severe disruption to the localized ROI overa short number of iterations.

Imaging microbes in an optically clear matrix

CyGEL (Biostatus) is an optically clear, thermoreversiblehydrogel mountant used for live cells. Here we used it tosuspend bacterial cells in 3D. A 50 μL mid-log bacterial cellsuspension was stained according to the BacLight protocol.The cells were washed in PBS (phosphate buffered saline) bycentrifugation and the pellet resuspended in 250 μL of icecold CyGEL. The CyGEL/cell suspension was applied to a glassbottom dish (MatTek) in a dome shape and brought to roomtemperature to gel.

A collagen matrix was made by mixing 50 μL of collagen(Kessler et al., 1960) with 50 μL LB (Luria Broth) and adjustedto pH 7.5 using 1.25 μL 0.8 M NaHCO3. The mix was appliedto a glass bottom dish and allowed to polymerize at roomtemperature. To incorporate microbes, 50 μL mid-log phasebacterial cell suspension in LB broth was added to the collagen,and the same protocol followed.

Imaging in all three cases was carried out using a C-Apochromat 40×/1.1 W Corr UV-VIS-IR objective and awavelength of 870 nm. Collagen second harmonic generatedsignals were detected using a 435–485 nm band pass emissionfilter. SYTO9 and PI were detected as previously described.

Results and discussion

Direct comparison between SP-LSCM and MP-LSM

To explore the quality of images of the bacteria we could obtainat different depths, E. coli TG1 18 was grown as a biofilm understatic conditions and stained with BACLight LIVE/DEAD. MP-LSM images were acquired using the 63×/1.4 oil, 40×/0.8water immersion and 20×/1.0 water dipping objectives. Asexpected, the 20×/1.0 water lens had the greater theoreticalworking distance and permitted deeper imaging but theachieved resolution was poor throughout compared to theother lenses, this was probably due to chromatic aberration inthe presence of the cover slip (note that the 20×/1.0 water lensis a dipping and not an immersion lens). The objective usedmost frequently was the 40×/0.8 water because its narrownose piece in conjunction with the original flow cell allowed aclose approach which was prohibited with the wider, relativelyflat topped lenses. The 63×/1.4 oil objective typically yieldedthe best results and readily enabled high-resolution images ofsingle cells to be clearly defined in x-, y- and z-dimensions,but the size of the nose-piece proved problematical with theoriginal flow cell.

MP-LSM was predicted to provide images to greater depththan SP-LSCM for two well-established reasons. Firstly, thelonger wavelength used for excitation is generally scatteredless and absorbed less, resulting in deeper penetration.Secondly, the excitation volume is significantly less in the z-plane compared to single photon. This enables the use of pin-hole free NDDs that in turn permit more scattered emission



Fig. 1. MP-LSM allows deeper imaging of a biofilm than SP-LSCM. Shown are z-slices of the same field of a BacLight LIVE/DEAD stained biofilm at theindicated depths into the biofilm; 0 μm is at the cover slip. The first column shows images obtained with single photon excitation with internal detectors,the second column with multiphoton excitation and with the same internal detectors and the third column with multiphoton excitation with specializedNDD detectors. Bar is 17 μm.

light to be collected. To test and illustrate the extent of thispredicted advantage for imaging biofilms we directly comparedimages obtained in the two modes.

Images of the same field obtained using the 40×/0.8 lenswith SP-LSCM or MP-LSM with the standard internal scanhead detectors and with the external NDDs are shown in Fig. 1.Measured depths are best approximations and should be takenas comparable measurements and not absolute as the effectof the biofilm on scatter and both chromatic and sphericalaberrations are unknown. The data show that at 0 μm allimaging modes can resolve individual bacteria. At 30 μm,all three approaches are near equal; however, by 60 μm thequality of the SP-LSCM images start to deteriorate significantlyand render some individual bacteria unidentifiable. At 90 μmMP-LSM using both internal and external detectors can still

yield decent images; however, the internal detector imagesare not as good as those obtained with the external NDDs.By 120 μm the internal detector configuration is unable toproduce satisfactory images whereas as the NDD configurationis still satisfactory and only by 180 μm is no longer able toproduce satisfactory images using the MP laser. Thus, usingMP-LSM in combination with external NDDs enabled studiesof individual bacteria up to 3× deeper compared to SP-LSCMand 1.5× deeper than MP-LSM using internal descanneddetectors. The images in Fig. 1 were obtained by applyingincreased laser power and detector voltage with increaseddepth, which is discussed in more detail below. Note thatto obtain these images it was important to optimize settingson the weakly fluorescent bacteria. This saturates the highlyfluorescent bacteria but enables all bacteria to be resolved. If


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the brighter bacteria are used to optimize the settings, the lowfluorescent bacteria appear as diffuse background and thusmislead results.

Identification of technical and biological variablesthat affect the biofilm image

As a result of extensive imaging, we have identified biologicaland technical variables that affect image quality. Here wediscuss these variables and the specific effects that changingvarious parameters or protocols have on image quality. Withinsight into the (dis)advantages of certain compromises, theresearcher can adjust the protocol to optimally suit the finalgoal of the imaging.

Choosing the fluorescent probe and optimal excitation and emissionwavelengths. Emission and excitation profiles for GFP, SYTO9and PI were generated. Based on these, we determined thesame excitation setting of 800 nm was suitable for all threefluorophores. This was combined with a 500–550 nm bandpass filter for green (GFP and SYTO9) emission and 575–640 nm band pass filter for red (PI) emission. The choiceof 800 nm allowed simultaneous excitation of both the redand green fluorescent substrates, but it should be noted thatusing the optimal excitation wavelength for an individualfluorescence marker could increase the depth of imaging(also see below) and overall image quality. However, if twofluorophores need to be visualized in the same sample asis the case for example with BACLight LIVE/DEAD, this inturn would require dual scanning and thus increase anyphototoxic effects. However, if for example only GFP is ofinterest, excitation at between 880–930 nm may be preferred.

The quality and depth of imaging was variable withthe emission wavelength of the fluorescent signal. Arepresentative example is shown in Fig. 1. At the sameexcitation wavelength, the signal associated with the longerwavelength emission, which was PI (red), was obtained togreater depth than the signal of the shorter wavelengthemission of SYTO9 (green). This is in accordance with physicalprinciples regarding light scatter, absorption and wavelength.However, if not taken into consideration, this variable couldin theory lead to an incorrect interpretation of BacLightLIVE/DEAD stained biofilms, where the red stain that is takenup by dead cells overrides the signal from the green dye. Thus,it could seem that all cells at significant depth were dead(red), whereas in reality technical enhancement would allowvisualization of the green dye and therefore visualization oflive (green) cells. This could be a particular concern if imagingis pushing the limits of sensitivity (as in Fig. 1, bottom panels).The recent development of the BacLight Redox Sensor CTCVitality Kit (Invitrogen) may allow further distinctions to bemade.

These effects of excitation and emission wavelength ofbiofilm images should also be taken into consideration when

choosing the fluorescent protein to use in imaging a biofilm.The longer excitation and emission wavelengths of tdTomatoor mCherry could be advantageous over GFP to image biofilmbacteria at greater depth. For most bacterial studies the useof mCherry could be preferable, due to its short maturationtime, good folding at 37◦C, high photostability and monomericnature. However, it is not as bright as tdTomato. That beingsaid, for photobleaching experiments a less stable variant likemStrawberry could be considered (Shaner et al., 2005). Withan ever-increasing range of available fluorescent proteins theeffect of the emission wavelength on imaging biofilms shouldbe taken into account as much as stability and maturationtime.

Optimizing image quality by adjusting power, gain and applyingfield averaging. The quality of the image at a given depthdepends not only on choosing the best excitation and emissionwavelength, but also on the amount of laser power applied.This can be adjusted as a percentage of the total output ofthe laser power. This value in turn will be variable for eachindividual wavelength, and depend on which lenses are used.Thus a more universal measure is to recalculate the percentagepower to absolute power applied, which is then expressed inWatts (also see ‘Materials and methods’).

In this study, laser power was always kept to a minimumto prevent biological damage and yet still yield good signal tonoise images. At increasing depths, laser power was increasedto off-set diminishing signal reaching the detector. This isdue to light scatter and to a lesser extent absorption of theexcitation laser light and specimen aberrations, which reducethe intensity at the point of focus with increased depth throughthe specimen. We applied this ramping of laser power in theimages in Fig. 1. Laser output was calibrated post-lens toapproximate applied power. This was only used as an estimateas the effects of the sample itself would prove highly variableon the laser power reaching the focal point. Specifically, forimages shown in Fig. 1 the multiphoton laser power wasramped from 10–350 mW. In the SP-LSCM images, at 488 nmthe power was ramped from 0.04 to 1.6 mW and at 561 nmfrom 0.12 to 1.5 mW. At 488 nm the laser power is 6 mW at100% transmission with 100% laser power, and at 561 nm thelaser power is 2 mW at 100% laser transmission. It should benoted that the multiphoton uses a pulsed laser and the actualphoton dose to the point of focus is significantly less than ifit were 350 mW of continuous wave light. Hence the laserpowers cannot be simply compared.

In conjunction with increased laser power, the detectorvoltage was also increased to help off-set the loss of emissionphotons due to increased scatter at the greater depth. Toachieve the same signal to noise by increasing laser poweralone would be damaging to the biological specimen, whichis demonstrated to our benefit below. Despite the increasednoise in the image as a result of increased detector gain, frameaveraging enabled data to be analysed. Frame averaging,



which uses multiple scans of the same field, was preferred overline averaging because it appears to permit better dissipationof any generated heat or accumulated free radicals and thusdecreased the chance of affecting the biological state of thebiofilm cells. The increased number of iterations requiredfor imaging a single field with frame averaging did notpresent a problem for viability under the conditions used (seebelow).

The actual increase in laser power that is required to obtainan optimal image will vary with excitation and emissionwavelength, fluorophore, laser and objective lens as well asthe biological characteristics of the site of imaging (see below).In summary, by increasing the laser power as well as thedetector gain and applying frame averaging, high-qualityimages can be obtained up to significant depth. Naturally,the detector voltage could be determined for the worse casescenario (i.e. at the greatest depth) to permit even less lightto be used at the near surface. However, this would resultin non-optimal images at the surface with a worse signal tonoise ratio than necessary and hence relatively poor imagesthroughout the entire z-stack not only at the greater depths.It was our preference to achieve the best images at all depthspossible without causing biological damage.

Biomass limits sensitivity. The E. coli strain formed biofilmsthat were highly structured, especially when grown in a flowcell, forming channels and mushrooms. When the biofilm wasimaged with the 40×/0.8 water objective it was noticed thatcells immediately adjacent to liquid, such as cells adjacentto a channel or at the edge of biofilm, could be visualizedat higher resolution with less laser power than was requiredfor obtaining comparable images of the adjacent field locatedwithin the biofilm. The most likely cause of this phenomenonis that the light path is optically clearer at the edge of thebiofilm where the mass is less dense compared to elsewhere inthe biofilm. This clearer optical path would result in decreasedscatter of the excitation and emission photons to and fromthe point of focus, and thus a clearer image with lower laserpower.

The hypothesis that increased light scatter due to biofilmbiomass alters the sensitivity limit was tested. Specifically,BacLight LIVE/DEAD stained cells were visualized in a threedimensional structure in the absence of significant biomasseither in a collagen matrix or in optically clear CyGEL(Biostatus). The collagen matrix was visualized using secondharmonics and the depth of penetration could be readilymeasured. In both cases, bacteria were visible up to the600 μm working distance, the limit of the 40×/1.1 waterobjective with a laser power of 125 mW. Where collagen wasused, the matrix material was also visible to this depth. Theimage clarity was consistently high throughout the z-stack(results not shown). This indicates that the depth limit weroutinely reached for a clear image of cells in a biofilm (120–150 μm) was restricted by the biofilm mass itself, increased

light scatter and possibly changes in refractive index. Biofilmbiomass composition varies significantly, changing withgrowth conditions and bacterial species or strain (Klausenet al., 2003b; Reisner et al., 2003; Wang et al., 2004; Goller& Romeo, 2008). Thus, it is important to appreciate thatthis effect of biofilm biomass on light scatter may result inlimits of detection that vary significantly in accordance withexperiment-specific biological parameters.

High-resolution time-lapse imaging of a growing biofilm in a flowcell. The specific type of flow cell used here has the advantagethat cover slips of high optical quality (#1.5) can be used tomaximize the benefit of the high NA objectives. However, toimage the biofilm in this flow cell with the MP-LSM requiredvarious adaptations to the hardware. A microscope stagemount was constructed to accommodate tubing and portsfrom the flow cell on the stage. This stage mount raised thesample and put it out of reach of the objective focal plane,so objective spacers were custom made to screw under theobjective in order to lift it closer to the specimen.

Furthermore, the viewing window in the standard model FC281 flow cell (Biosurface Technologies) was too narrow for thewider, high magnification, high NA objectives used and waswidened to allow use of the 63×/1.4 oil objective. A modelFC 81 flow cell has recently been modified with an even widerwindow to allow future use of the wider high NA objectives likethe 40×/1.1 water and the 63×/1.2 water. These objectivesmay confer advantages over the oil lens for certain biofilmsdepending on the apparent refractive index that may varywith composition of the matrix and cell packing. The choiceof the objective for prolonged time-lapse experiments in thisstudy was driven by the preference for an oil immersion lens,because water was subject to evaporation over an extendedperiod of time in the temperature controlled chamber setat 37◦C. Although a water immersion lens should performchromatically better than the oil lenses, the highly scatteringnature of the biofilm appeared to negate these effects andthe 63×/1.4 oil lens enabled improved imaging conditionsin comparison to all other lenses tested.

For time-lapse imaging of the biofilm with the 63×/1.4oil objective, the power level was set at 6 mW at 800 nm.Figure 2 shows the quality and detail of images that can beobtained using this hardware and protocols, in conjunctionwith the other advantages of MP-LSM. The rendered image ofa biofilm at 30 μm depth shows of a mix of E. coli expressingeither tdTomato or GFP. This illustrates that it will be possibleto examine single cells in the biofilm. To limit biologicalchanges it is important to keep a constant nutrient supply,and therefore we imaged with the pump on. However, withthis high magnification pulsing lines were seen across imagesas a result of the minor changes in fluid pressure. This wasresolved by scanning each field of view twice and applyingframe averaging after which these lines did not prevent furtheranalysis. However, it may be possible to synchronize image


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Fig. 2. High-resolution imaging of bacteria in a biofilm in a flow cell.A high resolution, rendered 3D image at 30 μm depth in a biofilm ofa mix of GFP- and tdTomato-expressing E. coli grown in the flow cell isshown. Image was obtained using a high magnification objective (Plan-Apochromat 63×/1.4 oil DIC). Bar is 5 μm.

collection and liquid flow if this problem still creates too muchimage distortion.

MP-LSM as a manipulation tool

In addition to an imaging tool, MP-LSM can be used as amanipulation tool, for example to achieve localized ablation,apoptosis/necrosis, bleaching or photoactivation of a well-defined volume of cells in the biofilm. With SP-LSCM this isnot possible to the same extent, because the excitation volumeextends through the z-plane to a much greater degree and isnot only confined to the focused ROI. Because of the confined x,y and z volumes achieved with MP-LSM excitation, prolongedor repeated imaging of the same field results in less biologicaldamage inflicted in the region outside the field of interest inthe sample compared to SP-LSCM. This can be important whenhigher excitation powers are used such as in bleaching andphotoactivation.

Establishing a viability baseline. To develop MP-LSM use formanipulating biofilms we first determined the amount ofimaging that can be tolerated on one field before bacterialdeath is obtained, as an indicator for extreme and undesirablebiological damage (Fig. 3). A biofilm was grown and BACLightLive/Dead stain added. One 225 μm2 region at a depth of30 μm was sequentially imaged for 800 frames using a laserpower of 45 mW at 800 nm with a 40×/0.8 water objective.Imaging took place for a period of 1315 s (800 sequentialiterations) with a 0.44 μm pixel dwell time of 2.56 μs. Foreach image, the green and red emitted fluorescence of the fieldwas quantified. The data show that there was no death withinthe first 250 iterations but that death became apparent overthe ensuing 125 and more iterations. This damage was mostlikely due to localized build up of free radicals and phototoxicdamage and possibly localized heating, both of which may benegated by increasing the time between scans at the point offocus, as happens by default when taking extended z-stacks.

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Fig. 3. The amount of imaging that can be tolerated on one field of abacterial biofilm before inducing cell death. One region was repeatedlyimaged in the presence of BACLight Live/Dead stain. The graph shows theincrease in propidium iodide intensity indicating death (thick line) over800 iterations at 1.5 s per frame. Also shown the intensity of SYTO9 stain(thin line) (see text).

Consistent with the data shown in Fig. 2, imaging a differentfield 310 times over a period of 320 s at 27 mW also did notinduce bacterial death. These experiments simply demonstratethe limits of continuous imaging which should be borne inmind for some experimental protocols.

Ablation, killing and bleaching of selected regions in a biofilm usingthe laser. A key variable to obtain either bleaching, killingor ablation is the level of laser power applied to the ROI.We identified parameters for ablating a small, defined ROIwithin a biofilm. A defined ‘hole’ was obtained by applying175 mW of laser power at 800 nm, to a ROI of 50×50 pixels(0.22 um/pixel) with a pixel dwell time of 1.28 μs and 200iterations over a 30 s time period (Fig. 4). This was immediatelyfollowed by an additional 140 s of imaging. The ablatedregion is indicated with an arrow in Fig. 4C. We presumethat the high laser power generates localized disturbance,again possibly due to localized heating, which on this occasionalso resulted in apparent localized water expulsion from theablated area affecting an area similar in size to the ablatedregion. Additional experiments showed that ablation withinthe centre of the biomass disrupted the structure of the biofilm,which also appeared to be due to forced liquid displacementas a result of heat dissipation. By contrast ablation of biomasson the edge of the biofilm minimized disruption of adjacentbiomass. The generated heat may also have contributed tothe cell death as judged by PI uptake. In the former case thiswas observed to occur immediately adjacent to the ablatedregion which became apparent within 60 s post-ablation. Byapplying a lower level of total laser power (125 mW at 800 nm,20 iterations) to a ROI than the laser power used to ablate,bacterial cell death could be induced as shown in Fig. 5. This



Fig. 4. Ablation of biofilm bacteria can be achieved using the laser. This time lapse shows the application of high laser power to a defined region (arrow)and the ensuing ablation of the cells in the region. The experiment was done in the presence of BACLight LIVE/DEAD stain. Bar is 20 μm. See text fordetails.

also further illustrates the use of the feature ROI, which wasdefined here in a heart shape, to identify the region within abiofilm to be manipulated.

Decreasing laser power further than required for killingshould result in photobleaching. This is essential for FLIP andFRAP (Fluorescence Loss In Photobleaching and FluorescenceRecovery After Photobleaching) type experiments (Lippincott-Schwartz et al., 2003; Koster et al., 2005), which arecommonly used as a tool on single cells to examine, for exampleprotein mobility (Phair & Misteli, 2000) or metabolic activity(Metcalf et al., 2008). By contrast, recovery of fluorescenceafter photobleaching of GFP-expressing bacteria in a biofilmcan be a result of migration of non-bleached bacteria tothe region or synthesis of new GFP. Thus, this can be usedto inform on bacterial motility in a biofilm or metabolicactivity of the cells. Using GFP-expressing cells, we successfullycaused photobleaching of the fluorescence in bacteria withina biofilm without inducing significant death using 50 mWpower with 200 iterations (Fig. 6). The exact combination ofpower and number of iterations that are required for thesedifferent applications will vary with the biological materialand location of the ROI in the biofilm. However, the examplesprovided here may serve as general guidelines and clearlyillustrate the potential of using MP-LSM for novel approaches

in the study of biofilms. Note that in Fig. 5, bleaching of thegreen fluorophore in the biofilm was not apparent presumablybecause, by contrast to the GFP, the (external) fluorophorewill continually diffuse in from the surrounding area. Bycombining this technology with, for example photoswitchablecyan fluorescent protein, these imaging techniques will enablemore intricate experiments to be performed such as trackingthe movement of individual bacteria in a biofilm.

The work described here complements and expands uponthe limited number of previous studies that illustrated generaland specific use of MP-LSM for analysis of bacterial biomass(Neu et al., 2004; Hu et al., 2005; Lacroix-Gueu et al.,2005; Briandet et al., 2008). These studies provide greaterguidance to researchers wishing to set up or optimize MP-LSMfor biofilm imaging. A range of bespoke hardware, confocalmethodologies and the optimal use of the microscope aredescribed in detail along with other biological and analyticalvariables that can significantly affect the image. We haveshown the potential benefits of MP-LSM over SP-LSCM thatwill allow significantly deeper imaging of single bacteria inbiofilms and multicolor 4D imaging. MP-LSM also enablesdetailed information to be gained on biofilm formation,structure and the dynamic behaviour of the cells in a biofilm.We suggest that the application of the laser power in the novel

Fig. 5. Small, specified regions of interest can be killed within a biofilm using the laser. This time lapse shows the application of ROI within a biofilm ofcells stained with BACLight LIVE/DEAD. Bar is 20 μm. The total power applied was adjusted to avoid ablation but sufficient to kill the selected bacteria(see text).


1 3 6 M . A . L A K I N S E T A L .

Fig. 6. A small, specified ROI can be bleached within a biofilm. This time lapse shows the application of ROI to bleach a small group of GFP-expressingcells within a biofilm in the presence of propidium iodide. Box indicates region bleached, and bar is 30 μm. Panel (A) situation prior to bleaching,(B) immediately following bleaching, (C) after 238 s (see text).

ways that are illustrated here can support the developmentof model systems to examine the effects of, for exampledrugs on individual cells in biofilm and on biofilm structureand regeneration. Combining an excellent understanding ofimaging technology and microbiology may thus lead to novelinsights into biofilm formation, dispersal and persistence.

Acknowledgements

We thank C. Beloin for the E. coli TG1 18 strain, and R. Leigh forassistance with the collagen matrix. This work was supportedby grant BB/F003692/1 from the BBSRC.

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