New trends in fluorescence in situ hybridization for identification and functional analyses of microbes
Graphical abstract
Highlights
► Double-labeled oligonucleotides – an easy boost for conventional FISH experiments. ► Highly parallel FISH detection of many taxa with new labeling and imaging strategy. ► FISH and transmission electron microscopy – from identity to ultrastructure. ► Padlock and polynucleotide probes detect genes in single microbial cells. ► New software and web-based tools for oligonucleotide and polynucleotide design.
Introduction
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 [1] 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 [2]. 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 [3].
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. These improvements and extensions of the FISH technique have been reviewed in detail [4, 5, 6, 7, 8, 9]. This review is intended to complement this information by providing a structured overview on the most recent developments in the FISH field by mainly focusing on publications, which appeared after 2008.
Section snippets
Advancements of conventional FISH techniques
The last years have seen important improvements of rRNA-targeted FISH techniques. For example, the group of Daniel Noguera has developed thermodynamically-based mathematical models of FISH that can now be easily utilized by FISH users for probe design and for in silico optimization of hybridization conditions via the web-based tool mathFISH [10•]. In addition, the introduction of 5′, 3′ doubly labeled oligonucleotide probes provides an easy means to increase the signal intensity of rRNA-FISH
New FISH techniques for gene detection
A major goal of microbial ecologists and medical microbiologists is to reliably identify microbes in complex sample material and to simultaneously obtain knowledge on the presence of genes in their genomes that are important for key physiological properties or encode virulence factors or antimicrobial resistance enzymes. FISH techniques are suitable to achieve this goal on a single-cell level and the last years have seen rapid progress in this field. Two basic approaches are applied for this
Conclusions and outlook
The last years have witnessed exciting improvements of the FISH technique, which have further improved its attractiveness for environmental and medical microbiologists. A thorough and easily accessible theoretical background for FISH has been established and new labeling and detection strategies offer increased sensitivity and have dramatically extended multiplexing options for the detection of microbes in their natural environment. These developments open a new window for system-level spatial
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We would like to thank David Berry and Alexander Loy for critical reading of the manuscript.
References (42)
- et al.
The identification of microorganisms by fluorescence in situ hybridisation
Curr Opin Biotechnol
(2001) - et al.
Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms
J Microbiol Methods
(2000) - et al.
Fluorescence in situ hybridisation for the identification and characterisation of prokaryotes
Curr Opin Microbiol
(2003) - et al.
Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays
Curr Opin Biotechnol
(2006) - et al.
Ciliate food vacuole content and bacterial community composition in the warm-monomictic crater Lake Alchichica (México)
FEMS Microbial Ecol
(2011) - et al.
A chemical method for fast and sensitive detection of DNA synthesis in vivo
Proc Natl Acad Sci USA
(2008) - et al.
Click chemistry as a reliable method for the high-density postsynthetic functionalization of alkyne-modified DNA
Org Lett
(2006) - et al.
Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells
Science
(1989) - et al.
Phylogenetic identification and in situ detection of individual microbial cells without cultivation
Microbiol Rev
(1995) - et al.
Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria
Appl Environ Microbiol
(2002)
Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques
Nat Rev Microbiol
Single-cell ecophysiology of microbes as revealed by Raman microspectroscopy or secondary ion mass spectrometry imaging
Annu Rev Microbiol
mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization
Appl Environ Microbiol
Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) improves signal intensity and increases rRNA accessibility
Appl Environ Microbiol
Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging
Proc Natl Acad Sci USA
daime, a novel image analysis program for microbial ecology and biofilm research
Environ Microbiol
Nitrite concentration influences the population structure of Nitrospira-like bacteria
Environ Microbiol
Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities
Environ Microbiol Rep
Ecophysiological analysis of microorganisms in complex microbial systems by combination of fluorescence in situ hybridization with extracellular staining techniques
Methods Mol Biol
Combination of fluorescence in situ hybridization with staining techniques for cell viability and accumulation of PHA and polyP in microorganisms in complex microbial systems
Methods Mol Biol
Single-cell analysis and isolation for microbiology and biotechnology: methods and applications
Appl Microbiol Biotechnol
Cited by (79)
Culturing the uncultured marine fungi in the omics age: Opportunities and challenges
2024, Fungal Biology ReviewsProspects for multi-omics in the microbial ecology of water engineering
2021, Water ResearchMolecular Biology Techniques for the Detection of Contaminants in Wastewater
2021, Wastewater Treatment: Cutting-Edge Molecular Tools, Techniques and Applied AspectsPursuing Human-Relevant Gut Microbiota-Immune Interactions
2019, ImmunityCitation Excerpt :Imaging techniques have been the most straightforward means for understanding the spatial distribution of microbes (Earle et al., 2015; Tropini et al., 2017). Fluorescence in situ hybridization (FISH) is a technique used to examine bacterial location within tissue samples and serves a critical tool in evaluating bacterial localization (Dejea et al., 2018; Swidsinski et al., 2005; Wagner and Haider, 2012). Mucosal biopsies and washings can also provide important information regarding mucosal associate communities, and new approaches that couple -omics technologies with co-localized elements in a microbiome provide a way to couple high-resolution functional analysis with spatial information (Sheth et al., 2019; Zmora et al., 2018).
Functional and molecular approaches for studying and controlling microbial communities in anaerobic digestion of organic waste: a review
2023, Reviews in Environmental Science and Biotechnology