Confocal Laser Scanning Microscopy and Digital Image Analysis
 | Fluorescence in situ hybridization (FISH) with rRNA-targeted probes provides unique possibilities to visualize uncultured microorganisms. FISH is also a powerful tool for analyzing microbial community structure. Probe-stained planktonic cells may be counted "by eye" in the epifluorescence microscope, but manual counting of tightly clustered cells in more complex samples is generally not feasible. Here the confocal laser scanning microscope comes to rescue,
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because it allows recording very sharp and clear images even of those labelled bacteria that live in aggregates and are embedded in thick and complex structures like biofilm. Computer algorithms can extract quantitative data from such high-quality confocal images.
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We are interested in adopting stereological principles in order to measure key features of microbial populations by confocal microscopy and digital image analysis. Recently, we implemented a method to obtain the biovolume fraction of bacteria detected by FISH (Schmid et al., 2000). A new quantitative FISH approach uses image analysis to determine not only the relative abundance, but also the absolute concentrations of clustered and heterogeneously distributed cells in complex samples (Daims et al., 2001). Currently we are analyzing the spatial localization of probe-stained bacteria, which have been captured in 3D confocal image stacks. For this purpose, the original 3D structure of a specimen must be preserved, and thus we have developed protocols to embed environmental samples in matrices that are well compatible with FISH and prevent sample deformation.
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Confocal image stacks are optimal input data for 3D visualization software. The resulting projections are useful to study the spatial arrangement of probe-stained microbial cells especially if the software provides arbitrary viewing perspectives.
Various 2D and 3D image analysis and visualization techniques are being implemented in our own software called DAIME (Digital image Analysis In Microbial Ecology). This program integrates urgently needed functionality to analyze confocal images of probe-stained single cells, cell aggregates, and filaments. Program features include 2D and 3D image segmentation, measurement of fluorescence brightness, surface area, absolute volume, and biovolume fraction of probe-defined populations, and interactive (close to real-time) volume rendering of confocal image stacks.  daime is under development and not yet suitable for general use, but will be available for download upon completion.
Investigated by: Holger Daims, Kilian Stoecker
Selected literature:
- Daims, H., Ramsing, N. B., Schleifer, K.-H. and Wagner, M. (2001). Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization. Appl. Environ. Microbiol. 67: 5810-5818.
- Schmid, M., Twachtmann, U., Klein, M., Strous, M., Juretschko, S., Jetten, M., Metzger, J. W., Schleifer, K.-H. and Wagner, M. (2000). Molecular evidence for a genus-level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation. System. Appl. Microbiol. 23: 93-106.
- Wagner, M., Horn, M. and Daims, H. (2002). Fluorescence in situ hybridization for the identification of prokaryotes. Curr. Opinion Microbiol. 6: 302-309.
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