Imaging biofilms using fluorescence in situ hybridization: seeing is believing

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.

Ecology of Legionella pneumophila biofilms: The link between transcriptional activity and the biphasic cycle

“…Nevertheless, the RNA extraction in biofilms may be the limiting factor when evaluating spatial organization due to the necessity to disrupt the biofilm structure. The extraction and amplification of genomic material fails to provide accurate insights into the morphological condition of the bacteria and the underlying three-dimensional microbial structures [ 34 , 166 ].…”

“…Allied with the technical progress of 3D imaging techniques, which are predominantly based on high resolution microscopy, such as confocal laser scanning microscopy (CLSM), fluorescence-based methods are employed to decode bacterial species patterns within biofilms at a single-cell and molecular levels [ 166 ]. These technologies include the expression of fluorescent proteins inserted into bacteria plasmids and fluorescence in situ hybridization (FISH) [ 34 ].…”

“…Nevertheless, these multiplex approaches can be limited to the number of bandpass filters used in imaging equipment's (the same happen to FP technique). However, several FISH variants may be used to increase the number of targets to dozens or hundreds [ 166 ]. In a FISH-based technique for combinatorial labeling and spectral imaging (CLASI-FISH), a unique combination of two or more fluorophores, selected to maximize spectral discrimination, are used in a same probe.…”

Microbial Symphony: Navigating the Intricacies of the Human Oral Microbiome and Its Impact on Health

“…Accurate detection and analysis are facilitated through the utilization of fluorophores that exhibit closely overlapping excitation and emission spectra. The combinatorial labeling approach entails the utilization of a preset repertoire of several fluorophores to selectively label a particular bacterium [ 98 , 99 , 100 , 101 ]. By implementing this combinatorial labeling technique, the range of distinguishable microbial taxa within a specific visual area is substantially increased [ 99 , 100 ].…”

The role of fungi in the diagnosis of colorectal cancer

“…Fluorescence in situ hybridisation (FISH) is a popular microbial detection technique (Liu et al 2021 ; Sampaio et al 2022 ). To detect intestinal mucosal microorganisms, CLAS-fish, BONCAT-FISH, HiPR-FISH, seq-FISH, and other techniques have been developed (Barbosa et al 2023 ). However, we need to develop new probes for a wider range of fungi (Petriglieri et al 2022 ).…”