The introduction of rRNA-targeted fluorescence in situ hybridization (FISH) using oligonucleotide probes for the cultivation-independent identification of microbes more than 20 years ago marked the beginning of a new era for environmental and medical microbiology. When integrated into the so-called full-cycle rRNA approach, FISH enables microbiologists to decipher complete structures of microbial communities in a quantitative manner. Furthermore, this phylogenetic staining technique in its basic format is easy to apply and once probes have been designed and evaluated, the detection of their target organisms in environmental or medical samples is straightforward and can be completed in a few hours. In its original format, fluorescent monolabeled oligonucleotide probes are used for FISH, but as the signal intensity of this technique is insufficient for cells with low ribosome contents, FISH detection efficiencies in oligotrophic environments are generally rather low. For such systems, catalyzed reporter deposition (CARD)-FISH, which exploits horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification is the method of choice to capture most microbial community members.
rRNA-targeting FISH techniques are continuously developed further and major improvements regarding increased cell permeability, accessibility of probe target sites, probe specificity, signal intensity, and so on have been achieved. A second rapidly evolving FISH-related research area is the combination of rRNA-FISH with other techniques, which provide additional information on (i) the presence of specific genes or mRNA molecules of the target cell, (ii) its specific metabolic activity or (iii) important environmental parameters such as the concentration of chemical compounds in the vicinity of the detected cell. For this purpose rRNA-FISH or CARD-FISH have been combined with various other FISH techniques and staining procedures as well as with microautoradiography, microelectrode measurements, Raman microspectroscopy, and NanoSIMS. This review provides an overview on the most recent developments in the FISH field.
New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr Opin Biotechnol. Nov 11 2011
Fluorescence in situ hybridization (FISH) has become an indispensable tool for rapid and direct single-cell identification of microbes by detecting signature regions in their rRNA molecules. Recent advances in this field include new web-based tools for assisting probe design and optimization of experimental conditions, easy-to-implement signal amplification strategies, innovative multiplexing approaches, and the combination of FISH with transmission electron microscopy or extracellular staining techniques. Further emerging developments focus on sorting FISH-identified cells for subsequent single-cell genomics and on the direct detection of specific genes within single microbial cells by advanced FISH techniques employing various strategies for massive signal amplification.