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The study of biofilms has elicited a lot of interest for the simple reason that biofilms are ubiquitous in nature. From a biotechnological standpoint, biofilms are major players in many production, fermentation and waste treatment processes involving immobilised cells. Biofilms in the environment determine water quality by influencing dissolved oxygen content and are involved in nutrient cycling or act as sink for toxic or hazardous materials. Biofilms are also the primary cause of dental plagues and infections in medical implants and contact lenses, which in some cases prove to be resistant to biocides.

Almost all microbial systems exist as biofilms or may be associated with them. Characklis and Marshall defined biofilms as "a surface accumulation, which is not necessarily uniform in time or space, that comprises cells immobilised at a substratum and frequently embedded in an organic polymer matrix of electrobial origin".

The application of confocal laser scanning microscopy (CLSM) has provided a powerful non-destructive method to biofilm research. CLSM application to biofilm work which began only in the early 90s, enabled complex microbial communities to be visualised in-situ revealing their most intricate architecture and organisation without disruption caused by fixation.

One major advantage of applying CLSM to biofilm study is its ability to obtain optical thin sections of fully hydrated biofilms. CLSM coupled with fluorescence methods and image analysis provides images with better resolution thus giving more information pertaining to biofilm composition. Data sets may be analysed to determine parameters such as cell numbers, cell area, orientation or even physiological processes such as diffusion co-efficients within biofilms, growth rates of micro-organisms, photosynthesis, enzymatic processes and even gene transfer activities. All of these can be visualised as three dimensional reconstruction, renderings or animations.

How do you observe biofilms?

Biofilm sample materials come in various forms and sizes. They can be observed directly from substrata where they were cultivated such as glass, polycarbonate or metal slides or other materials or in their natural environments such as rocks, leaves, wood or soil.

Biofilms in their natural habitats usually have irregular surfaces and thus need be mounted onto a regular surface such as a petri plate and then covered with their original liquid to prevent the biofilms from drying up prior to observation.

There are many staining options applied to biofilms. Fluorescent or reflective probes can be used depending on the sample. A wide range of nucleic acid stains such as the SYTO series (Molecular Probes), acridine orange, or 4',6-diamidino-2-phenylindole (DAPI) and gene probes tagged with fluorescein (FITC), rhodamine (TRITC), or cyanins (Cy2, Cy3, Cy5) have been used to observe micro-organisms.

Cell viability can also be demonstrated using specially designed fluorescent-based probes. The distribution and identification of specimens in complex biofilm communities have been determined with the application of fluorescent in-situ hybridisation (FISH) and fluorescently labelled antibody techniques.

Micro-organisms, particularly algae exhibit autofluorescence and hence add to the variety of colours imaged through a CLSM. Care must be taken though during interpretation of images as autofluorescent artefacts may be present in the specimens.

Extracellular polymeric substances (EPS) comprised of proteins, polysaccharides and nucleic acids represent an essential part as it provides the backbone of biofilms. In addition to nucleic acid stains, fluorescent stains that specifically bind to either proteins (Hoecsht 2495) or polysaccharides (Calcofluor White and Congo Red) are used. Antibodies labelled with fluorescent markers against protein work better than antibodies against polysaccharides.

Thus for polysaccharides, fluor-conjugated lectins have been extensively used in biofilms. Multiple staining with lectins can now be recorded simultaneously and thus specific lectin binding sites can be applied to provide more detail in the biofilm architecture.

Because a cocktail of compounds exists in biofilms, it may be worth looking at charge distribution in such a complex community. Fluor-conjugated dextrans have been used to determine charged residues as well as to track particle mobility in living biofilms.

Gene expression studies are likewise emerging interests in biofilm research using fluorogenic substrates and more recently, modified proteins such as the green fluorescent protein. Change in fluorescence in biofilms has also been used to monitor activities such as metabolic shifts as a result of chemical degradation or fluctuations in pH.

Microelectrodes aid in the detection of gradients occurring in biofilms. Although biofilms can be observed in their hydrated state, embedding techniques are advantageous for some specimens, particularly thick ones. Various embedding media are now available in the market, but the good old agarose gel is probably the simplest.

Considerations for confocal microscopy

Objective lenses play a pivotal role in getting the desired images. Greater confocality is obtained using high numerical aperture (NA) objective lenses (1.4).

The NA in conjunction with the pinhole size controls the thickness of an optical section. Imaging through thick samples (up to 1 mm) poses a problem to many microscopists but extra long working distance and some water-immersion lenses help solve this problem. The zoom function of the CLSM can also help in increasing resolution.

To obtain considerable detail in biofilm specimens, image collection is done using a filter such as the Kalman type mathematical filter that reduces the noise level of the image to get a clean image. This filter, however, may cause bleaching as a result of high frequency scanning.

Most manufacturers of CLSM use other filters primarily for image enhancement, to smooth the data sets and enhance the edges of objects within the image. Bio-Rad for example, offers low signal detection or photon counting mode to augment weak signals.

One advantage of CLSM is to present the image in 3D and this is done by scanning the specimen in the z direction. Bleaching effects during scanning resulting to signal loss can be compensated by manipulations in the instrument such as increase in the gain, which can be done manually or automatically.

As mentioned earlier, the NA determines thickness of the optical sections and thus the correct sectioning interval must also be considered to prevent over- or undersampling errors which are critical in the interpretation of the data. In certain instances, the use of antifade reagents may help delay photobleaching effects.

Multichannel imaging is now a standard option offered in CLSM, however, the combination of fluorochromes to be used must be carefully noted. Adjustments in the emission and excitation filter settings are also important to eliminate bleed through problems. Reflection imaging to visualise mineral and metal substances in biofilms is another function performed using CLSM systems.

More recently, multiphoton (MP) systems have been employed to reduce photobleaching and allow deeper penetration of excitation light in the absence of light obstructing pinholes. In an MP system, tunable femtosecond pulsed IR lasers are used to provide multiples of photons of longer wavelengths absorbed by lower wavelength fluorochromes.

Processing the images

Pictures of biofilm structures as revealed by electron microscopy appear to be flat and uninteresting. In contrast, the collected z series using CLSM can give rise to creative 3D reconstruction and animated presentations.

Pseudocolouring is also employed to colour code materials of the same type or same binding mechanism in biofilms to facilitate presentation and data analysis.

oftware packages included in the CLSM system have been mainly developed to quantify parameters captured as an image. It is now possible to get information from a certain region of interest or calculate cell area or numbers using the software packages such as Image Pro Plus or LaserPix. There is also free software on the internet such as NIH image or Image J for other image manipulations. With quantification techniques in place, it is exciting to know that biofilms grown under different conditions can now be quantitavely analysed and therefore compared.

Specific examples of biofilms in the environment

Microbial mats are classic examples of biofilms in the environment. They are associated with small solid/liquid interfaces and are affected by factors such as substratum quality, capillary attraction of water, light penetration, erosion rate and even grazing pressures. They can be found in intertidal flats, hypersaline coastal lagoons, alkaline lakes and hot springs. In most cases, the dominating micro-organisms are cyanobacteria, the colourless and purple sulphur bacteria and the sulphate reducing bacteria.

Photosynthesis and sulphur metabolism are the key activities occurring in microbial mats. CLSM has been used to visualise components in microbial mats in situ. Lotic systems characterised by the continuous movement of water are also major habitats of environmental biofilms. Streams, rivers or creeks are storage and transport avenues for a variety of materials.

Climate changes, topography, and geology, not to mention the indigenous macroorganisms living in the system affect lotic biofilms.

Both natural and simulated lotic biofilms have been studied extensively. CLSM has been used to follow biofilm development from the initial adhesion to detachment due to ageing or exposure to adverse conditions.

The effect of grazing by larger organisms present in the same environment has also been clearly demonstrated using CLSM.

Live bacteria were stained with SYTO 9 appearing as green and the EPS detected by TRITC-labelled Helix aspersa are shown in red.

Autofluorescece of cyanobacteria is presented as pink, while autofluorescence of algae is blue. Computer assisted quantification of biofilm components was done directly from the image. Characterisation of microbial communities on the basis of EPS produced is another technique that relies on the power of CLSM. This is specifically applied to river or lake snow or in limnologists' definition, aggregates that can be separated from water by a 0.45 µm filter.

FITC and TRITC labelled lectins were used to determine in-situ glycoconjugate distribution in these aggregates.

In situ staining of glycoconjugates at the polymer level was intended to complement in-situ hybridisation with rRNA targeted oligonucleotides at the cellular level. This could then be added to the microbial ecologists' toolbox of useful techniques.

Spatial distribution or location of metabolic activities can also be identified using CLSM. An example of such application is the in-situ analysis of nitrifying biofilms. The use of microelectrodes in combination with FISH pinpoints areas where ammonia or nitrite oxidation occurs. Consequently, the populations responsible for either ammonia or nitrite oxidation can be positively identified using specific gene probes and then visualised using a CLSM.

Looking ahead

With developments in fluorescence methods and innovations in microscopys, biofilm research has progressed considerably in the last five years. CLSM has provided researchers a means to look at biofilms such that their structures are preserved and their physiology uninterrupted. Degradation studies or bioremediation will definitely capitalise on the advantage of in-situ monitoring of microbial communities. With MP systems optimised, probing deep into the biofilms will most likely be a lesser challenge. The obstacle of accounting for viable but non-culturable organisms in the environment may finally be hurdled. Essentially, with CLSM, biofilms will be what you see is what you get!

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