Scanning Transmission X-Ray, Laser Scanning, and Transmission Electron Microscopy Mapping of the Exopolymeric Matrix of Microbial Biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Scanning transmission X-ray,laser scanning,and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.     

Nutrients and light effects on stream biofilms: a combined assessment with CLSM,structural and functional parameters

Nutrients and light are the most determinant factors for microbial benthic assemblages in oligotrophic forested streams. We investigated the importance of nutrients and light availability on the structure and the function of epilithic biofilms in a Mediterranean forested stream (Fuirosos, Spain). Biofilms grew on artificial substrata in both enriched and unenriched reaches where shade conditions were simulated. Four different treatments were generated: higher light unenriched, lower light unenriched, higher light enriched (HL-E) and lower light enriched. Chlorophyll a, bacterial density, extracellular polymeric substances (EPS), extracellular leucine aminopeptidase (LAmP) and alkaline phosphatase (APase) activities were analysed during the colonisation at days 4, 9, 16, 22 and 52. At day 52, confocal laser scanning microscopy (CLSM) was used to determine differences in biofilm architecture. CLSM evidenced differences in thickness and structural complexity of biofilms grown in different conditions. Biofilms in HL-E were the thickest and had the most complex structure. The CLSM highlighted that the EPS was agglomerated in the upper layer of enriched-grown biofilms, but evenly distributed through the biofilm in unenriched biofilms. CLSM 3D images suggested that cyanobacteria increased under higher nutrient conditions. Nutrient enrichment caused the decrease of APase activity. Interaction between the two factors affected LAmP activity. HL-E had the highest LAmP and the lowest APase activities, an indication that biofilm responses to nutrients mostly occurred with high-light availability. Our results revealed that the conjoint availability of light and nutrients caused the highest changes in biofilm spatial organisation, microbial structure and functioning in oligotrophic forested streams.

     

Depth Penetration and Detection of pH Gradients in Biofilms by Two-Photon Excitation Microscopy

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml · h⁻¹ with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 μm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.     

Concurrent Quantification of Cellular and Extracellular Components of Biofilms

Confocal laser scanning microscopy (CLSM) is a powerful tool for investigation of biofilms. Very few investigations have successfully quantified concurrent distribution of more than two components within biofilms because: 1) selection of fluorescent dyes having minimal spectral overlap is complicated, and 2) quantification of multiple fluorochromes poses a multifactorial problem. Objectives: Report a methodology to quantify and compare concurrent 3-dimensional distributions of three cellular/extracellular components of biofilms grown on relevant substrates. Methods: The method consists of distinct, interconnected steps involving biofilm growth, staining, CLSM imaging, biofilm structural analysis and visualization, and statistical analysis of structural parameters. Biofilms of Streptococcus mutans (strain UA159) were grown for 48 hr on sterile specimens of Point 4 and TPH³ resin composites. Specimens were subsequently immersed for 60 sec in either Biotène PBF (BIO) or Listerine Total Care (LTO) mouthwashes, or water (control group; n=5/group). Biofilms were stained with fluorochromes for extracellular polymeric substances, proteins and nucleic acids before imaging with CLSM. Biofilm structural parameters calculated using ISA3D image analysis software were biovolume and mean biofilm thickness. Mixed models statistical analyses compared structural parameters between mouthwash and control groups (SAS software; α=0.05). Volocity software permitted visualization of 3D distributions of overlaid biofilm components (fluorochromes). Results: Mouthwash BIO produced biofilm structures that differed significantly from the control (p<0.05) on both resin composites, whereas LTO did not produce differences (p>0.05) on either product. Conclusions: This methodology efficiently and successfully quantified and compared concurrent 3D distributions of three major components within S. mutans biofilms on relevant substrates, thus overcoming two challenges to simultaneous assessment of biofilm components. This method can also be used to determine the efficacy of antibacterial/antifouling agents against multiple biofilm components, as shown using mouthwashes. Furthermore, this method has broad application because it facilitates comparison of 3D structures/architecture of biofilms in a variety of disciplines.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.     

Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy.

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) was inoculated for 8 h at 50 ml. h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depths were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients developed. Microcolonies with pH values of below pH 3.0 were visible, in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 microm) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH within microbial biofilms.     

A two-step procedure for automatic and accurate segmentation of volumetric CLSM biofilm images

This paper presents a robust two-step segmentation procedure for the study of biofilm structure. Without user intervention, the procedure segments volumetric biofilm images generated by a confocal laser scanning microscopy (CLSM). This automated procedure implements an anisotropic diffusion filter as a preprocessing step and a 3D extension of the Otsu method for thresholding. Applying the anisotropic diffusion filter to even low-contrast CLSM images significantly improves the segmentation obtained with the 3D Otsu method. A comparison of the results for several CLSM data sets demonstrated that the accuracy of this procedure, unlike that of the objective threshold selection algorithm (OTS), is not affected by biofilm coverage levels and thus fills an important gap in developing a robust and objective segmenting procedure. The effectiveness of the present segmentation procedure is shown for CLSM images containing different bacterial strains. The image saturation handling capability of this procedure relaxes the constraints on user-selected gain and intensity settings of a CLSM. Therefore, this two-step procedure provides an automatic and accurate segmentation of biofilms that is independent of biofilm coverage levels and, in turn, lays a solid foundation for achieving objective analysis of biofilm structural parameters.     

Low-Load Compression Testing: a Novel Way of Measuring Biofilm Thickness

Biofilms are complex and dynamic communities of microorganisms that are studied in many fields due to their abundance and economic impact. Biofilm thickness is an important parameter in biofilm characterization. Current methods of measuring biofilm thicknesses have several limitations, including application, availability, and costs. Here, we present low-load compression testing (LLCT) as a new method for measuring biofilm thickness. With LLCT, biofilm thicknesses are measured during compression by inducing small loads, up to 5 Pa, corresponding to 0.1% deformation, making LLCT essentially a nondestructive technique. Comparison of the thicknesses of various bacterial and yeasts biofilms obtained by LLCT and by using confocal laser scanning microscopy (CLSM) resulted in the conclusion that CLSM underestimates the biofilm thickness due to poor penetration of different fluorescent dyes, especially through the thicker biofilms, whereas LLCT does not suffer from this thickness limitation.     

Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition

Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.     

Anticandidal Efficacy of Cinnamon Oil Against Planktonic and Biofilm Cultures of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida orthopsilosis</Emphasis>

Candida parapsilosis is yeast capable of forming biofilms on medical devices. Novel approaches for the prevention and eradication of the biofilms are desired. This study investigated the anticandidal activity of sixteen essential oils on planktonic and biofilm cultures of C. parapsilosis complex. We used molecular tools, enumeration of colony-forming units, the colourimetric MTT assay, scanning electron microscopy (SEM) and a chequerboard assay coupled with software analyses to evaluate the growth kinetics, architecture, inhibition and reduction in biofilms formed from environmental isolates of the Candida parapsilosis complex; further, we also evaluated whether essential oils would interact synergistically with amphotericin B to increase their anticandidal activities. Of the environmental C. parapsilosis isolates examined, C. parapsilosis and C. orthopsilosis were identified. Biofilm growth on polystyrene substrates peaked within 48 h, after which growth remained relatively stable up to 72 h, when it began to decline. Details of the architectural analysis assessed by SEM showed that C. parapsilosis complex formed less complex biofilms compared with C. albicans biofilms. The most active essential oil was cinnamon oil (CO), which showed anticandidal activity against C. orthopsilosis and C. parapsilosis in both suspension (minimum inhibitory concentration—MIC—250 and 500 μg/ml) and biofilm (minimum biofilm reduction concentration—MBRC—1,000 and 2,000 μg/ml) cultures. CO also inhibited biofilm formation (MBIC) at concentrations above 250 μg/ml for both species tested. However, synergism with amphotericin B was not observed. Thus, CO is a natural anticandidal agent that can be effectively utilised for the control of the yeasts tested.     

Biofilm structure differentiation based on multi-resolution analysis

Quantitative parameters for describing the morphology of biofilms are crucial towards establishing the influence of growing conditions on biofilm structure. Parameters used in earlier studies generally fail to differentiate complex three-dimensional structures. This article presents a novel approach of defining a parameter vector based on the energy signature of multi-resolution analysis, which was applied to differentiating biofilm structures from confocal laser scanning microscopy (CLSM) biofilm images. The parameter vector distinguished differences in the spatial arrangements of synthetic images. For real CLSM images, this parameter vector detected subtle differences in biofilm structure for three sample cases: (1) two adjacent images of a CLSM stack; (2) two partial stacks from the same CLSM stack with equal numbers of images but spatially offset by one image; and (3) three complete CLSM stacks from different bacterial strains. It was also observed that filtering the noise in CLSM images enhanced the sensitivity of the differentiation using our parameter vector.     

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions¹. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions^2,3.The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.     

Biofilm structure differentiation based on multi-resolution analysis

Quantitative parameters for describing the morphology of biofilms are crucial towards establishing the influence of growing conditions on biofilm structure. Parameters used in earlier studies generally fail to differentiate complex three-dimensional structures. This article presents a novel approach of defining a parameter vector based on the energy signature of multi-resolution analysis, which was applied to differentiating biofilm structures from confocal laser scanning microscopy (CLSM) biofilm images. The parameter vector distinguished differences in the spatial arrangements of synthetic images. For real CLSM images, this parameter vector detected subtle differences in biofilm structure for three sample cases: (1) two adjacent images of a CLSM stack; (2) two partial stacks from the same CLSM stack with equal numbers of images but spatially offset by one image; and (3) three complete CLSM stacks from different bacterial strains. It was also observed that filtering the noise in CLSM images enhanced the sensitivity of the differentiation using our parameter vector.     

In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms

Dermatophytes are fungi responsible for a disease known as dermatophytosis. Biofilms are sessile microbial communities surrounded by extracellular polymeric substances (EPS) with increased resistance to antimicrobial agents and host defenses. This paper describes, for the first time, the characteristics of Trichophyton rubrum and T. mentagrophytes biofilms. Biofilm formation was analyzed by light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) as well as by staining with crystal violet and safranin. Metabolic activity was determined using the XTT reduction assay. Both species were able to form mature biofilms in 72?h. T. rubrum biofilm produced more biomass and EPS and was denser than T. mentagrophytes biofilm. The SEM results demonstrated a coordinated network of hyphae in all directions, embedded within EPS in some areas. Research and characterization of biofilms formed by dermatophytes may contribute to the search of new drugs for the treatment of these mycoses and might inform future revisions with respect to the dose and duration of treatment of currently available antifungals.     

Biofilm formation on the surface of monazite and xenotime during bioleaching

Microbial attachment and biofilm formation is a ubiquitous behaviour of microorganisms and is the most crucial prerequisite of contact bioleaching. Monazite and xenotime are two commercially exploitable minerals containing rare earth elements (REEs). Bioleaching using phosphate solubilizing microorganisms is a green biotechnological approach for the extraction of REEs. In this study, microbial attachment and biofilm formation of Klebsiella aerogenes ATCC 13048 on the surface of these minerals were investigated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). In a batch culture system, K. aerogenes was able to attach and form biofilms on the surface of three phosphate minerals. The microscopy records showed three distinctive stages of biofilm development for K. aerogenes commencing with initial attachment to the surface occurring in the first minutes of microbial inoculation. This was followed by colonization of the surface and formation of a mature biofilm as the second distinguishable stage, with progression to dispersion as the final stage. The biofilm had a thin-layer structure. The colonization and biofilm formation were localized toward physical surface imperfections such as cracks, pits, grooves and dents. In comparison to monazite and xenotime crystals, a higher proportion of the surface of the high-grade monazite ore was covered by biofilm which could be due to its higher surface roughness. No selective attachment or colonization toward specific mineralogy or chemical composition of the minerals was detected. Finally, in contrast to abiotic leaching of control samples, microbial activity resulted in extensive microbial erosion on the high-grade monazite ore.     

Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.     

Micro-fabricated polydimethyl siloxane (PDMS) surfaces regulate the development of marine microbial biofilm communities

This study explored an antifouling (AF) concept based on deployment of microfabricated polydimethyl siloxane (PDMS) surfaces with 1–10?μm periodicity corrugated topographies in temperate marine waters. The effect of the surfaces on the development of microbial biofilms over 28?days and during different seasons, including both summer and winter, was examined using confocal laser scanning microscopy (CLSM) as well as terminal restriction fragment (T-RF) analysis for phylogenetic fingerprinting. The microscale topography significantly impacted biofilm development by altering the attachment pattern and reducing microcolony formation on the 1, 2 and 4?μm PDMS surfaces. Also, field deployments over 28?days showed a significant reduction in biovolume on the 4 and 10?μm PDMS surfaces despite altered environmental conditions. The microfabricated PDMS surfaces further significantly impacted on the community composition of the biofilms, as revealed by changes in T-RF profiles, at different stages of development. Moreover, altered biofilm resistance was demonstrated by exposing pre-established biofilms on 10?μm micro-fabricated surfaces to enhanced flagellate predation by a heterotrophic protist, Rhynchomonas nasuta. Pronounced changes in the overall marine microbial biofilm development as well as community composition warrant exploring substratum modification for marine AF applications.     

Investigation of the mesoscale structure and volumetric features of biofilms using optical coherence tomography

Optical coherence tomography (OCT) was successfully applied to visualize the mesoscale structure of three different heterotrophic biofilms. For this purpose, biofilm volumes of 4 × 4 × 1.6 mm³ were scanned with spatial resolutions lower than 20 µm within an acquisition time of 2 min. A heterogeneous structure was detected for biofilms cultivated in laminar as well as transient flow conditions. The structure was found to be more homogeneous for the biofilm grown in turbulent flow. This biofilm structure was characterized by a volumetric porosity of 0.36, whereas the porosity calculated for biofilms grown in laminar and transient conditions was 0.65. These results were directly generated from the distribution of porosity calculated from the OCT images acquired and can be linked to structural properties. Up to now, the mesoscale biofilm structure was only observable with time‐consuming and expensive studies, for example, magnetic resonance microscopy. OCT will most certainly be helpful for improved understanding and prediction of biofilm physics with respect to macroscale processes, for example, mass transfer and detachment as the information about mesoscale is easily accessible using this method. In the context of this study, we show that CLSM images do not necessarily provide an accurate representation of the biofilm structure at the mesoscale. Additionally, the typical characteristic parameters obtained from CLSM image stacks differ largely from those calculated from OCT images. Nevertheless, to determine the local distribution of biofilm constituents, microscopic methods such as confocal laser scanning microscopy are required. Biotechnol. Bioeng. 2010;107: 844–853. © 2010 Wiley Periodicals, Inc.