Computerized fluorescence microscopy of microbial cells

Abstract

The upgrading of fluorescence microscopy by the introduction of computer technologies has led to the creation of a new methodology, computerized fluorescence microscopy (CFM). CFM improves subjective visualization and combines it with objective quantitative analysis of the microscopic data. CFM has opened up two fundamentally new opportunities for studying microorganisms. The first is the quantitative measurement of the fluorescence parameters of the targeted fluorophores in association with certain structures of individual cells. The second is the expansion of the boundaries of visualization/resolution of intracellular components beyond the "diffraction limit" of light microscopy into the nanometer range. This enables to obtain unique information about the localization and dynamics of intracellular processes at the molecular level. The purpose of this review is to demonstrate the potential of CFM in the study of fundamental aspects of the structural and functional organization of microbial cells. The basics of computer processing and analysis of digital images are briefly described. The fluorescent molecules used in CFM with an emphasis on fluorescent proteins are characterized. The main methods of superresolution microscopy (nanoscopy) are presented. The capabilities of various CFM methods for exploring microbial cells at the subcellular level are illustrated by the examples of various studies on yeast and bacteria.