AUTHOR=Barbosa Ana , Miranda Sónia , Azevedo Nuno F. , Cerqueira Laura , Azevedo Andreia S. 

TITLE=Imaging biofilms using fluorescence in situ hybridization: seeing is believing

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 13 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1195803

DOI=10.3389/fcimb.2023.1195803

ISSN=2235-2988

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, will be explored, emphasizing their principal applications and how they have been addressed to improve the conventional FISH. Recent advances in fluorescence in situ hybridization (FISH)-based techniques (e.g., CLASI-FISH, BONCAT-FISH, HiPR-FISH) in combination with confocal laser scanning microscopy 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 approaches that will allow to dig deeper into the biofilm structure and function. 
The improvement and development of FISH-based approaches for an 3D identification and location of metabolites, genes and cells inside the biofilm without perturbing its structure in a cheap and easy way will be the major challenge in biofilm dynamics research.