Microscope-Based Imaging Platform for Large-Scale Analysis of OralBiofilms
L. Karygianni,a M. Follo,b E. Hellwig,a D. Burghardt,a M. Wolkewitz,c A. Anderson,a and A. Al-Ahmada
Department of Operative Dentistry and Periodontology, Albert Ludwigs University, Freiburg, Germany,a Department of Hematology and Oncology, Core Facility, AlbertLudwigs University, Freiburg, Germany,b and Institute of Medical Biometry and Medical Informatics, Albert Ludwigs University, Freiburg, Germanyc
A microscopic method for noninvasively monitoring oral biofilms at the macroscale was developed to describe the spatial distri-bution of biofilms of different bacterial composition on bovine enamel surfaces (BES). For this purpose, oral biofilm was grownin situ on BES that were fixed at approximal sites of individual upper jaw acrylic devices worn by a volunteer for 3 or 5 days. Eu-bacteria, Streptococcus spp., and Fusobacterium nucleatum were stained using specific fluorescence in situ hybridization (FISH)probes. The resulting fluorescence signals were subsequently tested by confocal laser scanning microscopy (CLSM) and moni-tored by an automated wide-field microscope-based imaging platform (Scan^R). Automated image processing and data analysiswere conducted by microscope-associated software and followed by statistical evaluation of the results. The full segmentation ofbiofilm images revealed a random distribution of bacteria across the entire area of the enamel surfaces examined. Significantdifferences in the composition of the microflora were recorded across individual as well as between different enamel surfacesvarying from sparsely colonized (47.26%) after 3 days to almost full surface coverage (84.45%) after 5 days. The enamel platesthat were positioned at the back or in the middle of the oral cavity were found to be more suitable for the examination of bio-films up to 3 days old. In conclusion, automated microscopy combined with the use of FISH can enable the efficient visualizationand meaningful quantification of bacterial composition over the entire sample surface. Due to the possibility of automation,Scan^R overcomes the technical limitations of conventional CLSM.
A common phenomenon in nature is the formation of dynamicsurface-adherent bacterial structures called biofilms (18, 33).
In the oral cavity in particular, multispecies oral biofilms consistof more than 700 species of bacteria, enmeshed in a polysaccha-ride-rich extracellular matrix (15, 25, 40). These specialized mi-crobial biofilms have evolved to endure in the adverse environ-ment of tooth surfaces and gingival epithelium (26, 32). Due tothis, their development is the outcome of numerous complexphysicochemical interactions between oral-tissue substrata, mi-croorganisms, and adsorbed macromolecules (16, 48). The initialcolonization of oral biofilms is characterized by the adherence oforal streptococci to pellicle proteins such as alpha-amylase, pro-line-rich proteins, and glycoproteins or to host receptors on epi-thelial cells (28, 43). Further oral biofilm formation can then beattributed either to coadhesion resulting from recognition be-tween planktonic cells and surface-bound cells or to coaggrega-tion due to cell-cell recognition between different species of bac-teria (26). Fusobacterium nucleatum coordinates the binding ofearly and late colonizers, particularly obligate anaerobes andstreptococci (21). In addition, bacteria interact with polysaccha-rides, mostly glucans, and other salivary proteins, contributing tothe maturation of dental plaque biofilms (23).
The biofilm research community has been showing an increas-ing interest in detecting and understanding the mechanisms ofbiofilm formation over the past decade. This microbiology-ori-ented focus was promoted by the fact that dental plaque biofilmshave been demonstrated to be crucial etiological factors in bio-film-mediated diseases such as caries, gingivitis, and periodontaldisease (11, 31, 46). The thin biofilm layer of Gram-positive mi-croorganisms that characterizes healthy patients is altered into achunky heterogeneous structure under the prevalence of numer-ous Gram-negative bacteria, usually observed in dental infections(27, 49). Complex interspecies interactions, including coaggrega-
tion, bacteriocin production, and metabolic and quorum-sensingcommunication among oral bacteria, are notable features of thismultispecies biofilm community. Bacterial biofilm communitiesalso present up to 1,000-times-higher resistance to antimicrobialagents, host immunity, and variations in other environmental fac-tors, such as pH and oxygen, than do their planktonic counter-parts (13, 47, 52). Therefore, the elimination of these persistentwell-structured bacterial ecosystems could play an integral role infinding efficient prevention and competent treatment strategiesagainst oral diseases.
The development of new methodologies for the visualizationof in situ- or in vivo-established biofilms has introduced a new erain this field of dental research (10, 20). Among the traditionalmethods used, viable plate count and culture-dependent tech-niques may inadvertently select for certain species in multispeciesbiofilms and can only poorly quantify semiplanktonic or desorbedbiofilms (29). High-resolution microscopic techniques such asscanning electron microscopy (SEM), environmental scanningelectron microscopy (ESEM), transmission electron microscopy(TEM), cryo-electron microscopy, and atomic force microscopy(AFM) make the ultrastructure of bacteria and their environmentvisible, but they cannot quantify adherent microorganisms (19).Among the more current bacterial identification techniques, thefluorescence in situ hybridization (FISH) method allows for the insitu study of the spatial and temporal dynamics of the bacterial
community by means of fluorescently labeled oligonucleotideprobes (5). The combined use of FISH with confocal laser scan-ning microscopy (CLSM) provides high-resolution three-dimen-sional (3D) images of individual microbial members in their nat-ural environment, but only over a limited sampling of the totalsurface area (1). Thus, CLSM monitors a specific confocal field,without taking into consideration the extensive discrepanciesamong various fields within the same biofilm (22). Optical ab-sorption and scattering also constrain the depth of optical pene-tration of CLSM, whereas unlabeled compounds and other spatialstructure features cannot be easily detected (44).
In view of the methodical restrictions involved in the estab-lished biofilm visualization techniques mentioned above, imageacquisition in our lab was done for the first time over an entiresample surface using a modular microscope-based screening sta-tion (Scan^R; Olympus Europa, Hamburg, Germany). We at-tempted to show how the Scan^R imaging platform based onhigh-content-screening microscope technology can fully auto-mate image processing and data analysis of high-content large-scale images, achieving a full segmentation of the image into sub-areas with evident biofilm formation. This report aims to describeand refine the characteristics of an experimental approach for theidentification of oral bacteria in intact in situ-grown biofilms bythe use of a Scan^R imaging platform combined with FISH. Forthis purpose, oral biofilms grown in vivo were examined for thepresence of Fusobacterium nucleatum and Streptococcus spp. usingspecies-specific FISH probes in addition to a general eubacterialprobe after 3 and 5 days, respectively. The results demonstrate thatthis visualization method is a promising technique for represen-tative analysis of oral biofilm composition.
MATERIALS AND METHODSSubjects and specimens. A healthy 33-year-old volunteer participated inthe study. Thorough clinical examination by an experienced dentist re-vealed good oral hygiene status. The volunteer abstained from antibiotics6 months prior to the beginning of the study. The study design was re-
viewed and approved by the Ethics Committee of the University ofFreiburg (proposal 222/08).
For the preparation of the specimens, the buccal surfaces of bovineincisors of 2-year-old cattle were removed and modified into cylindricalenamel specimens (diameter, 5 mm; 19.63-mm2 surface area; height, 1.5mm). Their bovine spongiform encephalopathy (BSE)-free status wasconfirmed after examination with the IDEXX Laboratories BSE diagnos-tic kit (Ludwigsburg, Germany). The enamel surfaces of all samples werethen polished by wet grinding with abrasive paper (400 to 4,000 grit). Theprotocol for disinfection of the enamel plates included ultrasonication inNaOCl (3%) for 3 min to remove the superficial smear layer, air drying,and ultrasonication in 70% ethanol for another 3 min. The disinfectedsamples were then ultrasonicated twice in double-distilled water for 10min and, finally, stored in distilled water for 24 h to hydrate prior toexposure in the oral cavity (1).
Red wax was used to fix the enamel specimens on the approximal sidesof an individual upper-jaw acrylic appliance over periods of 3 and 5 days.Six disinfected enamel slabs were placed in the interdental area betweenupper premolars and molars, so that the movements of the tongue orcheek could not inhibit biofilm formation (Fig. 1). The visualization andquantification of the adherent bacteria were achieved by combined use ofFISH with the Scan^R screening microscope (2). For each time period,the volunteer carried six enamel samples that were rinsed off with sterile0.9% saline solution for 10 s after their exposure in the oral cavity.
FISH. FISH was carried out according to a modified protocol for theenamel slabs, previously described by Amann et al. and modified by Al-Ahmad et al. (3, 5). In brief, after removal from the oral cavity, the bio-films formed on the enamel slabs were fixed in 4% paraformaldehyde inphosphate-buffered saline (PBS; 1.7 mM KH2PO4, 5 mM Na2HPO4 with0.15 M sodium chloride [pH 7.2]) for 16 h at 4°C. The specimens werethen rinsed off twice with sterile PBS and fixed again in a solution com-posed of ethanol (50%) in PBS (1:1, vol/vol) for 12 h at 4°C. The probeswere subsequently washed twice with PBS and permeabilized in a solutioncomprising 7 mg/ml of lysozyme (hen egg white lysozyme; Fluka, Buchs,Switzerland; 105,000 U/mg in 0.1 M Tris-HCl, 5 mM EDTA [pH 7.2]) for9 min at 37°C in a humid chamber. Thereafter, the specimens were rinsedoff twice with PBS and dehydrated in an ascending series of ethanol con-centrations (50 to 100%) for 3 min each.
The hybridization of the enamel slabs was conducted in 24-well plates(Greiner Bio-One, Frickenhausen, Germany), wrapped with aluminumfoil, at 46°C for 90 min. Each well contained 600 �l hybridization buffer (5M NaCl, 1 M Tris-HCl [pH 8.0], 25% [vol/vol] formamide, and 10%[wt/vol] sodium dodecyl sulfate [SDS]) and 1 �l oligonucleotide probefor eubacteria, fusobacteria, and streptococci (50 ng/�l). The hybridiza-tion of the specimens followed their incubation in 600 �l wash buffer (159mM NaCl, 20 mM Tris-HCl [pH 7.5], 5 mM EDTA [pH 8.0], and 0.01%[wt/vol] SDS) per well for 15 min at 48°C.
High-pressure liquid chromatography (HPLC)-purified oligonucleo-tide probes for eubacteria, Streptococcus spp., and F. nucleatum were com-mercially synthesized and 5=-end labeled (Thermo Fisher ScientificGmbH, Ulm, Germany). The specificity of the oligonucleotide probes(EUB 338, FUS 664, and STR 405) listed in Table 1 was tested previouslyusing different bacterial strains (3).
CLSM. All FISH probes were excited at the following wavelengths:fluorescein, 488 nm; Cy3, 546 nm; and Cy5, 633 nm. The measurement ofthe fluorescence emission of the probes was carried out at the followingwavelengths: fluorescein, 495 to 565 nm; Cy3, 552 to 592 nm; and Cy5, 649
FIG 1 Individual upper-jaw acrylic appliance with the enamel slabs in place atdifferent locations. The specimens were positioned at the front (f), in themiddle (m), and at the back (b), on both sides, right (R) and left (L), of theappliance. The exposed surfaces were fixed toward the tooth enamel by redwax.
TABLE 1 Sequences, 5= modifications, target species, and references for used oligonucleotide probes
to 703 nm. Further analysis of the labeled biofilms was conducted byCLSM (Leica TCS SP2 AOBS) by placing the specimens face down onto adrop of physiological saline solution in a chambered coverslip (�-Slide, 8well; Ibidi, Germany) and examining them with the aid of a 63� waterimmersion objective (HCX PL APO/bd.BL 63.0 � 1.2 W; Leica). Sequen-tial scanning was used to minimize the risk of spectral overlap between theprobes (2). In brief, three representative locations on the enamel plateswere chosen for the visualization of the oral biofilm. The upper and lowerboundaries of the biofilm at each location were determined, and the meanthickness of the biofilm was calculated using these measurements fromthe three locations. Biofilms were scanned in the Z-direction at these threepoints, producing sections of a thickness of approximately 0.5 �m each at2-�m intervals throughout the biofilm layers. Each image was taken witha resolution of 1,024 by 1,024 pixels and a zoom setting of 1.7, correspond-ing to physical dimensions of 140 by 140 �m.
Automated microscopy. (i) Sample preparation and instrument cal-ibration. After the in situ hybridization of the enamel probes, the speci-mens were covered in a mixture of 20 �l Peakflow TM beads (Blue flowcytometry reference beads; Invitrogen, Germany) and PBS (1:10 [vol/10vol]). The samples were then left to dry in the dark for 1 h at roomtemperature. The blue beads were detected using a 4=,6-diamidino-2-phenylindole (DAPI) filter set (excitation, 352 nm to 402 nm; emission,437 nm to 475 nm). These beads were used to enable autofocusing of themicroscope, whether or not biofilm was present at a particular location(Fig. 2). The samples were then placed face down in a chambered coverslipas for the confocal microscopy experiments.
(ii) Image acquisition. Image acquisition was conducted using a 20�objective (UPLSAPO N.A. 0.75) on a Scan^R high-content-screening sys-tem based on an IX-81 inverse microscope stage (Olympus Europe, Ham-burg, Germany). The excitation of the probes was carried out with theMT20 illumination system (xenon-mercury burner, 150 mW; Olympus,Germany). For measurement of the FISH fluorescence, the following filtersets were used: fluorescein isothiocyanate (FITC) (excitation, 494 to 504nm; emission, 534 to 549 nm), Cy3 (excitation, 540 to 550 nm; emission,583 to 594 nm), and Cy5 (excitation, 620 to 650 nm; emission, 700 to 737nm).
In order to determine the location of the outer boundaries of thesamples, three representative locations were chosen along the edge of eachenamel slab with the aid of the Scan^R software. Each of the 12 slabs wasscanned at up to 177 nonoverlapping positions, covering the entire sur-face of the plate, in such a way that each image was completely containedwithin the area of the plate. Each of these images was derived from Z-stacks made up of 10 sections taken at intervals of 2 �m and constitutinga total imaging depth of 20 �m. Because we were interested in the platebiofilm coverage rather than the three-dimensional (3D) structure of thebiofilm, these images were then combined as maximal projections at eachof the positions and for each of the channels.
(iii) Image analysis. Each image consisted of 1,346 by 1,024 pixels andcovered a total area of 433 by 330 �m. Within the Scan^R analysis pro-gram, every image in turn was subdivided into 475 virtual subobjects of 50by 50 pixels or approximately 16 by 16 �m each; these were then used asthe smallest units of measurement for the subsequent data analysis.Therefore, each enamel slab had a total number of up to 84,075 data
points which could be used for measurement. The goal was to try and lookglobally at the sample, rather than capture individual bacteria. The thresh-olding was carried out on these virtual subobjects through the use of dotplots depicting mean FITC signal in the Scan^R software and then en-closed within a region, similar to that used in typical flow cytometry anal-yses. The subobjects which were contained within this region were con-sidered to be positive for the eubacterial probe and were controlled againby referencing them to the original images. The number of subobjects wasthen used to calculate the total amount of subobjects containing eubacte-ria. Similarly, the mean intensities of Cy3 and Cy5 were also used todetermine whether or not a region was positive for these probes; however,in both of these cases, the subobjects had to also be positive for the FITCsignal in order to minimize the occurrence of any possible false-positivesubobjects from the Cy3 and Cy5 channels. A background correction wasperformed for each channel, and the presence or absence of biofilm wasdetermined by setting intensity thresholds for each of the three probes(eubacteria, Streptococcus spp., and F. nucleatum), which were then usedacross the entire sample. The evaluated regions with positive fluorescencesignals for eubacteria (FITC) were used to determine the 100% value ofthe bacterial biofilm on the plate and therefore to determine the coveragelevel of the samples in percentages. For a subobject to be considered pos-itive for Streptococcus spp. or F. nucleatum, it had to also be positive foreubacteria. The area covered by these target bacterial cells was then mea-sured as a percentage of the FITC-positive regions.
Statistical analysis. For the descriptive exploration of the data, boxplots were calculated and graphically displayed, stratified by side, posi-tion, and time. The coefficient of variation was calculated stratified byside, position, and time. An analysis of variance (ANOVA) was used. Foreach examined subject (eubacteria, Streptococcus spp., and F. nucleatum),the continuous response variable was modeled as a function of side, po-sition, and the corresponding interaction as explanatory variables, andseparately for time. Model assumptions were graphically checked by re-siduals and other regression diagnostics (including Cook’s distance). Thenormality of error terms was assumed. All calculations were done usingPROC MIXED from the statistical software SAS 9.1.2.
RESULTSCLSM. Representative images of biofilm formation are presentedin Fig. 3. After FISH, the visualization of the bacterial biofilms onthe enamel surfaces was tested by confocal laser scanning micros-copy (CLSM). All bacterial species examined were detectable onthe enamel slabs after exposure in the oral cavity for 3 days. Themorphology of the majority of the microorganisms was coccoid,but filamentous bacteria of various configurations were also iden-tified. Inter- and intraindividual variety was manifested upon ob-servation of single aggregates, mono- or multilayered bacterialchains, and three-dimensional bacterial clumps of various sizes.
Automated microscopy. (i) Quantitative analysis of biofilmformation after 3 days. Figure 4 shows a FISH-Scan^R tiled im-age paired with the graphical distributions of the different regionswhich were positive for the respective bacterial targets (eubacteria,Streptococcus spp., and F. nucleatum) of a 3-day-old oral biofilmgrown in situ on enamel slabs.
Three days after biofilm formation, 47.26% of the enamel sur-face was coated with bacteria. The covering grades of the substra-tum by Streptococcus spp. and Fusobacterium nucleatum were19.73% and 3.45% after 3 days, respectively. Statistical analysisshowed significant differences (P � 0.0337) in the contents ofeubacteria and Streptococcus spp. on the enamel surfaces locatedon the left (31.38% and 17.71%) and right (63.15% and 21.24%)sides of the oral cavity, respectively. Differences between the twosides were not significant for F. nucleatum (P � 0.1752). Addition-ally, the bacterial content seemed to vary depending on the posi-
FIG 2 Cross-section of oral biofilm on bovine enamel slabs. Blue referencebeads were applied on the biofilm surface for instrument setup and calibration.
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tion of the enamel slabs in the oral cavity. Less biofilm was formedon the specimens situated in the middle (25.55%) than on thefront (53.04%) and posterior (63.15%) specimens (P � 0.0.0001).The posterior samples also had higher contents of Streptococcusspp. and F. nucleatum (42.5% and 6.87%) than the front (11.06%and 2.05%) and middle (5.1% and 1.12%) specimens, respectively(P � 0.0001).
(ii) Quantitative analysis of biofilm formation after 5 days. InFig. 5, tiled overviews of images derived from the scanning ofbiofilm-coated enamel plates hybridized with three different fluo-rescence probes are presented. The quantitative results of the bac-teria detected are also shown in Fig. 6 in the form of box plots.
Five days after biofilm formation, 84.45% of the enamel sur-face was covered with microorganisms. Streptococcus spp. and Fu-sobacterium nucleatum showed covering grades of 49.37% and15.98% after 5 days, respectively. Statistical differences (P �0.00003) in the coverage of eubacteria and Streptococcus spp. weredetected between the left (87.59% and 55.16%) and the right(81.59% and 43.58%) specimens in the oral cavity, respectively.Differences between the two sides were not significant for F. nu-cleatum (P � 0.8309). As far as the bacterial content in differentpositions of the samples in the oral cavity is concerned, a higherpercentage of eubacteria was detected on the front (87.64%) spec-imens than on the middle (82.8%) and on the posterior (83.31%)samples (P � 0.0332). The posterior samples also had a highercontent of Streptococcus spp. (56.65%) than those positioned atthe front (47.83%) or in the middle (43.63%) of the oral cavity(P � 0.0001). F. nucleatum was mainly located on the middle(20.28%) and posterior (20.66%) surfaces and less on the front(7%) ones (P � 0.0001). The percentages presented above are allmedian values.
DISCUSSION
For the first time, the current study has established a minimallydestructive scanning approach to examine oral biofilm composi-tion across entire sample surfaces by the combined use of FISHwith 16S rRNA-targeted oligonucleotide probes and automatedmicroscopy. This enabled an overall assessment of oral bacterialdiversity and distribution across the enamel slabs. The perceptionof biofilms as landscapes is one of the latest trends in oral biofilm
research, focusing on the large-scale distribution and movementsof microorganisms in the framework of microbial biogeography(8). Indeed, understanding the role of microorganisms in oraldiseases such as dental caries, gingivitis, and periodontitis neces-sitates a thorough visualization of the spatial distribution of apredominant species (7, 54). In addition to this, a clear vision ofthe entire biofilm structure unravels the underlying mechanismsof spatial biofilm patterning in relation to biofilm development.Numerous factors which affect biofilm formation and develop-ment, such as complex interspecies interactions, differences inlocal substrate concentrations or other nutrients, and environ-mental changes (pH and oxygen), can be highlighted throughlarge-scale image observation (8). In their study, Nielsen et al.investigated a flow cell-grown model consisting of Burkholderiasp. strain LB 400 and Pseudomonas sp. strain B13 (FR1) whichinteracted metabolically (37). With the aid of the FISH-CLSMtechnique, they successfully monitored a shift in the spatial struc-ture of the consortium, after a shift from noncommensal to com-mensal conditions. Furthermore, the outcomes of antimicrobialtreatment on oral biofilms often involve structural alterations ac-companied by functional changes within the biofilms. In a studyby Hope and Wilson, the influences of 0.05 and 0.2% (wt/vol)chlorhexidine (CHX) were visualized by CLSM on multispeciesbiofilms, modeling interproximal plaque grown on a hydroxy-apatite substrate (24). Image analysis revealed biofilm contractionat a rate of 1.176 �m�1 after exposure to 0.2% CHX.
The use of bovine enamel slabs provides a representative modelfor the study of bacterial colonization on surfaces. A great numberof easily gained, homogeneous bovine enamel surfaces were usedin this study due to their common physicochemical propertieswith human dental hard tissues. Past studies have indicated thatthere are no detectable differences between the compositions ofthe biofilms formed on artificial and natural tooth surfaces (38),whereas there are some evident discrepancies in the compositionof the pellicle (39).
A parameter of great relevance, the covering grade of the entireenamel surface, has been examined in this study. The number ofmicroorganisms on the enamel slabs increased significantly withthe oral exposure time. Thus, the total proportions of Streptococ-cus spp. and F. nucleatum were 23.18% and 65.35% of the total
FIG 3 FISH-CLSM micrographs of enamel slabs after 3-day in situ exposure in the oral cavity to control the specificity of FISH probes. Green, eubacteria (a);yellow, F. nucleatum (b); magenta, Streptococcus spp. (c).
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bacteria after 3 and 5 days, respectively. This corresponds roughlyto the values for vital bacteria as reported by Arweiler et al. (6).Coaggregation plays a key role in bacterial colonization of thetooth surface (29). This is especially important for Streptococcusspp. and F. nucleatum. FISH studies conducted at time intervals oflonger than 72 h may reveal interactions between the differentbacterial species (41). A CLSM study by Dige et al. demonstratedthe predominance of streptococci in biofilm during the first 6 to48 h (14). Streptococci secrete the extracellular enzyme glycosyl-transferase. In saliva and in the pellicle, three isoforms of thisenzyme are usually detected: glycosyltransferases B, C, and D (51).These enzymes synthesize specific receptors for the attachment ofstreptococci, which have a relatively short doubling time (9). Theabove factors could offer an explanation for the dominance ofstreptococci in the first days of dental plaque maturation. How-ever, the presence of streptococci might be overemphasized be-cause the culture methods used may select for certain species (3).
Fusobacterium nucleatum, a Gram-negative microorganism,has the ability to coaggregate with early and late colonizers (45).Therefore, F. nucleatum has an extremely important function as abridge among oral bacteria. It is considered to be a relevant initi-ator of periodontal disease resulting from matured subgingivalplaque. Guggenheim et al. studied a biofilm formed in vitro andconsisting of five species (17). After 2.5 days, F. nucleatum wasdetected at a percentage of 50%. In our study, F. nucleatum rep-resented only a small proportion of the initially adhering bacteriathat was influenced by the exposure time of the enamel slabs. After5 days, F. nucleatum represented a greater part (15.98%) of theadherent bacteria than after 3 days (3.45%), and it was mainlysituated on the middle and posterior samples. A possible explana-tion could involve proband-specific characteristics and the pres-ence of higher oxygen concentrations at the frontal sites of the oralcavity, since the presence of the late colonizer F. nucleatum isbound to low oxygen concentration (17).
FIG 4 FISH-Scan^R image and graphical distribution of different biofilm bacteria (eubacteria, Streptococcus spp., and F. nucleatum) of a 3-day-old oral biofilmgrown in situ on enamel slabs. The image seen in panel A is a tiled overview of the microbial colonization pattern showing an overlay of all three staining patterns.Oral biofilm was stained simultaneously with the all-bacterium-specific EUB 338 probe (green), the Streptococcus-specific EUB 405 probe (red), and the F.nucleatum-specific FUS 664 probe (blue). Panels B, C, and D graphically depict the spatial distribution of positive subobjects after thresholding and analysis inthe Scan^R analysis program, containing eubacteria, Streptococcus spp., and F. nucleatum and labeled with FITC, Cy3, and Cy5, respectively. x and y axis valuesrepresent the different positions on the microscope stage.
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The results of the present study showed some very significantdifferences in the local distribution of Streptococcus spp. and F.nucleatum among the different specimens. The enamel slabs thatwere positioned at the back or in the middle of the oral cavity haveproven to be more suitable for the examination of biofilms up to 3days old. In contrast, the front enamel slabs would be indicated forstudies in which biofilms older than 5 days are involved. Previousreports showed no influence of the position of the specimens onthe data (3). However, the current results are more representativedue to the greater number of measuring points obtained with theautomated microscope. Further interpretation implies differencesin oxygen supply of the samples, the availability of saliva, and thesaliva flow rate, as well as differences in tooth anatomy.
There is currently an increasing demand for proper quantifi-cation of the highly variable phenotypes found in oral biofilms.The subjectivity due to a low number of representative images canbe avoided only by conducting a higher number of objective mea-surements of biofilm composition. Therefore, automated micros-copy incorporates automated image acquisition of multistainedoral bacteria and image analysis of these data facilitating auto-mated scanning of entire surfaces. The automated determinationof the focal plane for each field of view is a crucial parameter foracquiring stable and reproducible biofilm images. Low-qualityscreenings of irrelevant biofilm components with intense fluores-cence signal or of only a small number of oral microorganismsusually result from focusing either on the entire field of view or onthe most eye-catching plane containing visible structures (30). Forthis reason, we decided to use fluorescent microbeads added to thesample after biofilm formation to identify the most appropriatefocal plane, independently of whether bacterial biofilm was pres-ent or not at a given location. The use of a 20� objective wasdeemed to be sufficient for our purposes. It allowed the acquisi-tion of images covering the entire area of interest on the enamelslabs with enough detail to be informative, but at the same timeallowing the scans to be completed within a reasonable amount oftime and without a drying out of the substrate. Another criticalfactor concerning image acquisition is the autofluorescence of thesubstrate. After a number of pilot studies in which the backgroundof enamel autofluorescence was minimal, we carried out our ex-periments with natural tooth surfaces, despite the fact that in someinstances autofluorescence has been shown to impede the inter-pretation of fluorescence signals of in situ biofilms (53).
In order to obtain robust large-scale images, efficient staining
of the different oral microbial species and reproducible image ac-quisition were essential prerequisites. The bacterial populations ofinterest constituting the biofilms could be quantified and com-pared after the application of fluorescence in situ hybridization(FISH). The FISH protocol applied in this study, in which theoligonucleotide probes were labeled with FITC, Cy3, or Cy5, de-livered specific fluorescence signals from eubacteria, Streptococcusspp., or F. nucleatum, respectively, and the signals could be easilydistinguished after merging images from the different color chan-nels. Some of the most important advantages of FISH for the iden-tification of bacteria in oral biofilms are the ability to detect un-cultured bacteria and the rapid availability of new oligonucleotideprobes. However, the restrictions of this method are caused by thefact that the specific oligonucleotide FISH probes can stain onlyintact ribosomes in vital bacteria. Ribosomes in dead bacterialcells are degraded quickly due to loss of membrane integrity (5).Finally, stable illumination and sensitive detection of the emittedfluorescence during the excitation of the oligonucleotide probeswere taken into consideration as supplementary factors affectingthe perceived image quality (35).
For the automated image analysis after background correction,the positive regions for each of the fluorescent probes were iden-tified via a combination of thresholding based on fluorescenceintensity and gating. These thresholds were applied to all of theimages from a single plate. To our knowledge, there has been nosoftware solution to overcome the technical restrictions concern-ing automatic recognition of highly diverse bacterial populations.Some open-source software packages such as CellProfiler Analyst/Cell Classifier and EBImage constitute fast image-processing en-gines capable of classification by machine learning of cell recogni-tion, but they are not optimal for use with the irregular structuresnormally encountered in biofilms (12). The development of newsoftware specialized in the detection of bacterial cells and biofilmstructures could automate the control of a whole range of fluores-cence-based imaging assays in microbiology, since high-content-screening platforms such as Scan^R are computer controlled andfully motorized (12).
The scanning of a single enamel slab (5 mm in diameter) asdescribed in this report took approximately 2 h to complete. Atfirst glance, this could be considered too time-consuming, espe-cially compared with relatively faster visualization techniquessuch as CLSM. However, the ability of automated microscopy tomeasure large-scale biofilm development across an entire surface
FIG 5 Overviews of tiled images constructed from the individual Scan^R maximum-projection images of the supporting enamel surface of representative5-day-old in situ oral biofilms on bovine enamel plates. Oral biofilm was hybridized with three different specific probes (EUB 338, FUS 664, and STR 405). Shownare results obtained with green eubacterium-specific probe (EUB 338) (a), red Streptococcus-specific probe (STR 405) (b), and blue Fusobacterium nucleatum-specific probe (FUS 664) (c) after 5 days and all three probes after 5 days (d). Scale bars, 20 �m.
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FIG 6 Box plots depicting percentages of the different bacterial members in 3- and 5-day-old oral biofilm as detected by FISH. The central line is the median;whiskers indicate minimum and maximum. Each box indicates the front (f), middle (m), or back (b) position of the enamel slabs on the right (r) and left (l) sidesof the oral cavity, respectively.
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area with high spatial resolution has its own advantages. In theconfocal microscopy experiments, 3 positions were chosen “ran-domly” within a plate, with each position covering a region of 140�m by 140 �m. In contrast, the Scan^R autofocus allowed“hands-off” scanning, which was able to follow surface irregular-ities across an entire plate, in addition to the ability to tile thepositions of the image stacks. The circular shape of the platesallowed us to use a “3-point method” to set the boundaries foreach plate, enabling the entire surface area of the plate to bescanned automatically. Scanning an entire plate brings a level ofobjectivity that is difficult to obtain when choosing positions byhand, however carefully it is done. The automated microscopyapproach also allowed analysis of the entire plate as a unit, ratherthan having to examine multiple individual stacks. Covering amuch larger surface than is practical to do manually allowed us togather much more information per plate, leading to improvedstatistical analysis.
Magnetic resonance imaging (MRI) would appear to be an-other option for noninvasive visualization of bacterial structureswith various applications on dental biofilms. Mapping of struc-tural biofilm characteristics, biofilm structure/flow relation, andflow and transport properties as well as diffusion profiles consti-tutes information demonstrated in MRI time-resolved studies. Ina recent study, minimally destructive monitoring of biofilm pro-cesses was conducted by combining an integrated nuclear mag-netic resonance (NMR) system with a confocal laser scanning mi-croscope. The entire three-dimensional microscopic structure ofthe biofilm was successfully visualized by MRI, while complemen-tary fluorescence information obtained by CLSM increased ourknowledge about oral biofilms in a depth-resolved fashion (34).At first glance, MRI appears to be suitable for mapping the spatialcharacteristics of most kinds of oral biofilms. Although this maybe true for in vitro-grown monospecies biofilms thicker than 200�m, a challenge is posed by the significantly thinner in situ-grownmultispecies oral biofilms. The technical restrictions of thismethod are highlighted by the lack of contrast in the density of 1Hnuclei between the microbial biomass and the bulk phase, espe-cially if we consider that most oral biofilms consist of more than95% water (36). Unless additional contrast is applied to the im-ages, they are of poor quality and unsuitable for further analysis.The relatively low resolution of the device—approximately 7 to 15�m—is definitely a factor contributing to the failure of this tech-nique to efficiently scan and monitor dental biofilms thinner than200 �m. On the other hand, automated microscopy offers higherresolution, which allows full mapping of the locations of the bac-terial communities present and, combined with FISH detection,enables the specific and simultaneous identification of multiplebacterial species within the biofilm.
From an ecosystematic point of view, variations in oral bio-films can be detected only if a representative area of the biofilm isanalyzed. The automated microscopy-based system used in thisstudy allows the minimally destructive investigation of intact bio-film across the complete surface of the enamel plate. The Scan^Rhas the ability to produce and further analyze multiple thin sec-tions of the biofilm (30). In contrast, the CLSM technique man-ages to analyze only a restricted amount of data due to a limitednumber of available measuring points. The Scan^R software en-ables the quantification of various bacterial populations and theirmetabolic activities within the oral biofilm. The measured fluores-cence intensity then correlates with the metabolic activity of the
bacteria within the biofilm. Alterations in bacterial metabolic ac-tivity could be caused by different dental materials, nutrient com-ponents, and antibacterial mouthwashes.
To our knowledge, there have been no studies to date whichhave conducted large screenings of oral biofilm composition.Only the study of McLean et al. (34) managed to visualize in vitro-grown single-species biofilm using nuclear magnetic resonanceand confocal microscopy. However, this technique is not appro-priate for distinguishing different bacterial species within a mul-tispecies biofilm. Moreover, the automated microscopy techniquepresented here has already been used for the visualization of largenumbers of eukaryotic cells (12). In the present study, automatedmicroscopy proved to be an efficient method for the screening oflarge-scale in situ-grown oral biofilms. Until now, the existingreports have described visualization techniques with the aid ofconfocal microscopy to examine representative areas of the oralbiofilm. Future studies should compare this automated micros-copy-based method with the conventional confocal microscopymethod which has been used to date.
In conclusion, fluorescence labeling techniques such as FISHare applicable to the evaluation of bacterial adherence to enamelsurfaces. Therefore, the screening ability of automated micros-copy, combined with the use of FISH, enables the efficient visual-ization and meaningful quantification of bacterial populationsacross an entire sample surface. In the future, the same method-ological approach should be applied to more individuals in orderto investigate more bacterial species, as well as to study biofilmgrowth on other materials such as composites, fiberglass posts,and implant materials.
ACKNOWLEDGMENTS
Gabi Braun is acknowledged for skillful technical laboratory assistanceduring FISH.
This study was supported by the German Research Foundation (DFG,AL 1179/1-1).
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