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The objective of the current review is to summarize the current state of optical methods in redox biology. It consists of two parts, the first is dedicated to genetically encoded fluorescent indicators and the second to Raman spectroscopy. In the first part, we provide a detailed classification of the currently available redox biosensors based on their target analytes. We thoroughly discuss the main architecture types of these proteins, the underlying engineering strategies for their development, the biochemical properties of existing tools and their advantages and disadvantages from a practical point of view. Particular attention is paid to fluorescence lifetime imaging microscopy as a possible readout technique, since it is less prone to certain artifacts than traditional intensiometric measurements. In the second part, the characteristic Raman peaks of the most important redox intermediates are listed, and examples of how this knowledge can be implemented in biological studies are given. This part covers such fields as estimation of the redox states and concentrations of Fe–S clusters, cytochromes, other heme-containing proteins, oxidative derivatives of thiols, lipids, and nucleotides. Finally, we touch on the issue of multiparameter imaging, in which biosensors are combined with other visualization methods for simultaneous assessment of several cellular parameters. [Display omitted] • Detailed classification of genetically encoded fluorescent redox biosensors. • Redox biosensors and fluorescence lifetime imaging microscopy (FLIM). • Raman spectroscopy for redox biology. [ABSTRACT FROM AUTHOR]

Abstract A wide variety of genetically encoded fluorescent biosensors are available to date. Some of them have already contributed significantly to our understanding of biological processes occurring at cellular and organismal levels. Using such an approach, outstanding success has been achieved in the field of redox biology. The probes allowed researchers to observe, for the first time, the dynamics of important redox parameters in vivo during embryogenesis, aging, the inflammatory response, the pathogenesis of various diseases, and many other processes. Given the differences in the readout and spectra of the probes, they can be used in multiparameter imaging in which several processes are monitored simultaneously in the cell. Intracellular processes form an extensive network of interactions. For example, redox changes are often accompanied by changes in many other biochemical reactions related to cellular metabolism and signaling. Therefore, multiparameter imaging can provide important information concerning the temporal and spatial relationship of various signaling and metabolic processes. In this review, we will describe the main types of genetically encoded biosensors, the most frequently used readout, and their use in multiplexed imaging mode. Graphical abstract fx1 Highlights • Genetically encoded indicators: main principles and architectural types. • Different readout types of genetically encoded indicators. • Examples of studies using redox indicators in multiplexed imaging mode. [ABSTRACT FROM AUTHOR]