A significant number of genetically encoded indicators based on fluorescent proteins that allow detecting changes in various parameters: membrane potential shift, pH, concentrations of hydrogen peroxide, lactate, pyruvate, NAD+/NADH, ATP, calcium cations, etc. have been created. Some of them (for example, indicators of calcium cations and hydrogen peroxide) are successfully used by numerous groups of researchers in experiments in vivo. The fundamental limitation of most fluorescent indicators is their ability to only qualitatively assess the change in the parameter of interest in the cell. The nature of this limitation is that it is difficult to compare the absolute value of the measured parameter and the detected fluorescent signal for various reasons. Quantitative measurements impose an additional requirement on the indicator signal: it should not depend on the technical features of the experiment (such as the level of protein expression, the type and power of the excitation source, a set of fluorescent filters, etc.). The fulfillment of this requirement may be associated with the detection of the indicator signal in the time domain, where the main characteristic of the signal is the fluorescence lifetime. The fluorescence lifetime is an absolute value, insensitive to changes in the intensity of the excitation source, detection settings, losses in the optical path or sample, changes in the concentration of the fluorophore in the sample, photobleaching. Thus, when calibrating an indicator in the time domain, a specific value of the measured parameter is associated with a specific value of the fluorescence lifetime. The time-resolved imaging mode imposes its own limitations on the properties of the indicator's fluorescent core. Thus, the lifetime of its fluorescence should change in proportion to the value of the measured parameter, which is not characteristic of any fluorophore. In this work, we make a brief review of the principles of detection of the fluorescent signal of genetically encoded indicators, focusing on current approaches related to the analysis of time-resolved data.
Keywords: FLIM; fluorescence lifetime; fluorescent indicators.