Single-Cell Imaging and Sequencing-Based Detection of Microorganisms Using Highly Sensitive Fluorescence in situ Hybridization (FISH)

Abstract

Fluorescence in situ hybridization (FISH) has become a standard technique in the visual detection and phylogenetic identification of environmental microorganisms in marine microbiology. Thirty years have passed since the appearance of FISH, and now FISH can detect not only ribosomal RNA but also mRNA and functional genes with high sensitivity. This chapter describes the principles, drawbacks, and applications of FISH and highly sensitive FISH. In particular, we introduce two high-sensitivity FISH methods developed by the authors. The first is in situ DNA hybridization chain reaction (HCR), a highly sensitive and highly penetrating detection technique for ribosomal-RNA–targeting FISH, and the second is two-pass tyramide signal amplification (TSA)-FISH that can detect single-copy genes. Finally, we discuss how the FISH technique has contributed to the field of marine microbiology.

Keywords: CARD-FISH, Environmental microbiology, FISH, Functional genes, GeneFISH, HCR-FISH, Highly sensitive FISH, in situ DNA-HCR, ISH, Marine bacteria, Microscopic observation, Oligonucleotide probe, Phylogenetic identification, Polynucleotide probe, Probe permeabilization, Single cell imaging, Two-pass TSA-FISH, Visual detection.