New trends in fluorescence in situ hybridization for identification and functional analyses of microbes

https://doi.org/10.1016/j.copbio.2011.10.010Get rights and content

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.

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)

  • R. Amann et al.

    Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques

    Nat Rev Microbiol

    (2008)
  • M. Wagner

    Single-cell ecophysiology of microbes as revealed by Raman microspectroscopy or secondary ion mass spectrometry imaging

    Annu Rev Microbiol

    (2009)
  • L.S. Yilmaz et al.

    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

    (2011)
  • K. Stoecker et al.

    Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) improves signal intensity and increases rRNA accessibility

    Appl Environ Microbiol

    (2010)
  • A.M. Valm et al.

    Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging

    Proc Natl Acad Sci USA

    (2011)
  • H. Daims et al.

    daime, a novel image analysis program for microbial ecology and biofilm research

    Environ Microbiol

    (2006)
  • F. Maixner et al.

    Nitrite concentration influences the population structure of Nitrospira-like bacteria

    Environ Microbiol

    (2006)
  • B. Knierim et al.

    Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities

    Environ Microbiol Rep

    (2011)
  • J.L. Nielsen et al.

    Ecophysiological analysis of microorganisms in complex microbial systems by combination of fluorescence in situ hybridization with extracellular staining techniques

    Methods Mol Biol

    (2010)
  • J.L. Nielsen et al.

    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

    (2010)
  • S. Ishii et al.

    Single-cell analysis and isolation for microbiology and biotechnology: methods and applications

    Appl Microbiol Biotechnol

    (2010)
  • Cited by (79)

    • Molecular Biology Techniques for the Detection of Contaminants in Wastewater

      2021, Wastewater Treatment: Cutting-Edge Molecular Tools, Techniques and Applied Aspects
    • Pursuing Human-Relevant Gut Microbiota-Immune Interactions

      2019, Immunity
      Citation 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).

    View all citing articles on Scopus
    View full text