LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Porto, Portugal.
ALiCE - Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.
Front Cell Infect Microbiol. 2023 May 22;13:1195803. doi: 10.3389/fcimb.2023.1195803. eCollection 2023.
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 hybridization (FISH) has been arguably the most widely used method for an 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.
生物膜是一种复杂的结构，其中居住的微生物、细胞外基质和周围环境之间存在着复杂的关系。由于生物膜在医疗、环境和工业等如此多样化的领域无处不在，因此人们对生物膜的兴趣呈指数级增长。分子技术（例如，下一代测序、RNA 测序）已被用于研究生物膜特性。然而，这些技术会破坏生物膜的空间结构；因此，它们不允许观察生物膜成分（例如细胞、基因、代谢物）的位置/位置，这对于探索和研究微生物的相互作用和功能尤为重要。荧光原位杂交 (FISH) 可以说是最广泛用于分析生物膜空间分布的方法。在这篇综述中，我们将探讨已经应用于生物膜研究的不同 FISH 变体（例如 CLASI-FISH、BONCAT-FISH、HiPR-FISH、seq-FISH）。与共聚焦激光扫描显微镜结合使用，这些变体成为可视化、定量和定位生物膜内微生物、基因和代谢物的强大方法。最后，我们讨论了开发基于 FISH 的稳健和准确方法的新的可能研究方向，这些方法将使我们能够更深入地研究生物膜的结构和功能。
