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Imaging biofilms using fluorescence in situ hybridization: seeing is believing

Barbosa, Ana, Miranda, Sónia, Azevedo, Nuno F., Cerqueira, Laura, Azevedo, Andreia S.·2023·Frontiers in Cellular and Infection Microbiology
DOI 10.3389/fcimb.2023.1195803PMC PMC10239779
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Abstract

Biofilms are complex structures with an intricate relationship between the resident microorganisms, the extracellular matrix, and the surrounding environment. Interest in biofilms is growing exponentially given its ubiquity in so diverse fields such as healthcare, environmental and industry. Molecular techniques (e.g., next-generation sequencing, RNA-seq) have been used to study biofilm properties. However, these techniques disrupt the spatial structure of biofilms; therefore, they do not allow to observe the location/position of biofilm components (e.g., cells, genes, metabolites), which is particularly relevant to explore and study the interactions and functions of microorganisms. Fluorescence in situ hybridization (FISH) has been arguably the most widely used method for an in situ analysis of spatial distribution of biofilms. In this review, an overview on different FISH variants already applied on biofilm studies (e.g., CLASI-FISH, BONCAT-FISH, HiPR-FISH, seq-FISH) will be explored. In combination with confocal laser scanning microscopy, these variants emerged as a powerful approach to visualize, quantify and locate microorganisms, genes, and metabolites inside biofilms. Finally, we discuss new possible research directions for the development of robust and accurate FISH-based approaches that will allow to dig deeper into the biofilm structure and function.

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Figure 1 Steps of traditional FISH protocol (I). First, the biofilm sample is fixed to stabilize the cells and permeabilize the cell membrane (1). Then, labelled probes are added and allowed to hybridize with the rRNA target (2), and the excess probe is washed away (3). Finally, the sample is analyzed under epifluorescence microcopy or confocal laser scanning microscopy to determine the spatial distribution of biofilm populations (4). Species in polymicrobial biofilms can be organize in three different ways (II): (A) separate microcolonies (confocal microscopy image reprinted with permission from (Mark Welch et al., 2016)); (B) co-aggregation (confocal microscopy image reprinted with permission from (Azevedo et al., 2016)); (C) arranged in layers (confocal microscopy image reprinted with permission from (Almeida et al., 2011)). Created with BioRender.com.
Figure 1 Steps of traditional FISH protocol (I). First, the biofilm sample is fixed to stabilize the cells and permeabilize the cell membrane (1). Then, labelled probes are added and allowed to hybridize with the rRNA target (2), and the excess probe is washed away (3). Finally, the sample is analyzed under epifluorescence microcopy or confocal laser scanning microscopy to determine the spatial distribution of biofilm populations (4). Species in polymicrobial biofilms can be organize in three different ways (II): (A) separate microcolonies (confocal microscopy image reprinted with permission from (Mark Welch et al., 2016)); (B) co-aggregation (confocal microscopy image reprinted with permission from (Azevedo et al., 2016)); (C) arranged in layers (confocal microscopy image reprinted with permission from (Almeida et al., 2011)). Created with BioRender.com.CC BY · PubMed Central
Figure 2 Principle steps of (A) CARD-FISH, (B) NAM-FISH and (C) FIVH. The four steps of traditional FISH are the basic standpoints of these modified FISH variants, being highlighted in the figure the main improvements made on the hybridization step. (A) In direct CARD-FISH, horseradish peroxidase (HRP) conjugated probes hybridized with the complementary rRNA sequence, and signal amplification is conducted with fluorescein labeled tyramides; on the other hand, in the indirect CARD-FISH, biotinylated probes and HRP-labeled streptavidin are used; (B) In NAM-FISH panel is showed the chemical structure of the most used NAMs (LNA, PNA and 2’OMe); (C) In FIVH protocol, the fixation and permeabilization step is eliminated and the NAM probe is given by oral gavage; then, the hybridization occurs inside of mouse body, at normotemperature (37°C), and the visualization of fluorescent cells can be performed directly using a confocal laser endomicroscope. Created with BioRender.com.
Figure 2 Principle steps of (A) CARD-FISH, (B) NAM-FISH and (C) FIVH. The four steps of traditional FISH are the basic standpoints of these modified FISH variants, being highlighted in the figure the main improvements made on the hybridization step. (A) In direct CARD-FISH, horseradish peroxidase (HRP) conjugated probes hybridized with the complementary rRNA sequence, and signal amplification is conducted with fluorescein labeled tyramides; on the other hand, in the indirect CARD-FISH, biotinylated probes and HRP-labeled streptavidin are used; (B) In NAM-FISH panel is showed the chemical structure of the most used NAMs (LNA, PNA and 2’OMe); (C) In FIVH protocol, the fixation and permeabilization step is eliminated and the NAM probe is given by oral gavage; then, the hybridization occurs inside of mouse body, at normotemperature (37°C), and the visualization of fluorescent cells can be performed directly using a confocal laser endomicroscope. Created with BioRender.com.CC BY · PubMed Central
Figure 3 Schematic representation of type of probes used in (A) DOPE-FISH and (B) CLASI-FISH. The 4 main basic steps of traditional FISH protocol are also common to these FISH variants. (A) DOPE-FISH approach uses oligonucleotide probes that are double-labeled with different fluorophores at their 5′- and 3′-end, allowing a detection up to 7 microorganisms; (B) CLASI-FISH, microorganisms are hybridized with two or more fluorochrome conjugated probe. Using 8 fluorochromes in this combinatorial labeling approach, it is possible to create up to 28 specific combinations. Created with BioRender.com.
Figure 3 Schematic representation of type of probes used in (A) DOPE-FISH and (B) CLASI-FISH. The 4 main basic steps of traditional FISH protocol are also common to these FISH variants. (A) DOPE-FISH approach uses oligonucleotide probes that are double-labeled with different fluorophores at their 5′- and 3′-end, allowing a detection up to 7 microorganisms; (B) CLASI-FISH, microorganisms are hybridized with two or more fluorochrome conjugated probe. Using 8 fluorochromes in this combinatorial labeling approach, it is possible to create up to 28 specific combinations. Created with BioRender.com.CC BY · PubMed Central
Figure 4 Schematic representation of (A) MAR-FISH, (B) NanoSims-FISH and (C) BONCAT-FISH. The 4 main basic steps of traditional FISH protocol are also common to these FISH variants. (A) MAR-FISH and NanoSims-FISH are based on the incorporation of radiolabelled compounds and hybridization of a fluorescently labeled rRNA-targeted probe to examine the phylogenetic identity and the metabolic activity of cells. In MAR-FISH, the FISH and MAR signal is visualized simultaneously by a CLSM. For NanoSIMS-FISH, the hybridized cells are analyzed by CLSM and subsequently the analysis of metabolic activity is obtained in a NanoSIMS instrument. (C) In BONCAT-FISH approach, the BONCAT assay (in vivo incorporation of the non-canonical amino acid L-azidohomoalanine; green cells in figure) is combined with a fluorescently labeled rRNA-targeted probes (FISH assay; red cells in figure), and the cells are analyzed/visualized by CLSM. Created with BioRender.com.
Figure 4 Schematic representation of (A) MAR-FISH, (B) NanoSims-FISH and (C) BONCAT-FISH. The 4 main basic steps of traditional FISH protocol are also common to these FISH variants. (A) MAR-FISH and NanoSims-FISH are based on the incorporation of radiolabelled compounds and hybridization of a fluorescently labeled rRNA-targeted probe to examine the phylogenetic identity and the metabolic activity of cells. In MAR-FISH, the FISH and MAR signal is visualized simultaneously by a CLSM. For NanoSIMS-FISH, the hybridized cells are analyzed by CLSM and subsequently the analysis of metabolic activity is obtained in a NanoSIMS instrument. (C) In BONCAT-FISH approach, the BONCAT assay (in vivo incorporation of the non-canonical amino acid L-azidohomoalanine; green cells in figure) is combined with a fluorescently labeled rRNA-targeted probes (FISH assay; red cells in figure), and the cells are analyzed/visualized by CLSM. Created with BioRender.com.CC BY · PubMed Central

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Metadata

Authors
Barbosa, A., Miranda, S., Azevedo, N. F., Cerqueira, L., & Azevedo, A. S.
Venue
Frontiers in Cellular and Infection Microbiology
Publisher
Frontiers Media SA
Published
2023-05-22
Volume
13
ISSN
2235-2988
DOI
10.3389/fcimb.2023.1195803
PMC
PMC10239779
Cited by
63
References
5
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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CoChat. (2026). Citation check: Imaging biofilms using fluorescence in situ hybridization: seeing is believing. https://app.cochat.ai/papers/10.3389/fcimb.2023.1195803
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Barbosa, A., Miranda, S., Azevedo, N. F., Cerqueira, L., & Azevedo, A. S. (2023). Imaging biofilms using fluorescence in situ hybridization: seeing is believing. Frontiers in Cellular and Infection Microbiology. https://doi.org/10.3389/fcimb.2023.1195803