ИСТИНА

The use of nonlinear optical techniques for the study of various biological tissues in vivo has an avalanche-like dynamic that is associated both with the development of laser technology and the expansion of the range of new protein sensors. A noticeable volume of investigations targeted on the in vivo monitoring of reactive oxygen species (ROS) with a cellular (or even sub-cellular) resolution in optically transparent biological models (Caenorhabditis elegans, Danio rerio and Xenopus laevis larvae) has been performed using wide-field and confocal fluorescence microscopy techniques. A switch to the study of redox signaling in rodents, a closer model to humans, is difficult because of the strong scattering of the tissue in the optical range. One solution of the task is the implantation of special optical fibers for excitation and reading the indicators of ROS. This method has been successfully applied in our group to study the dynamics of acidosis in the deep layers of the cortex and caudate nucleus of the rat brain during ischemic stroke and reperfusion [1]. Sub-cellular spatial resolution at depths of up to 1 mm in the biotissues could be provided by multi-photon interrogation of fluorescent sensors. In this work, we have developed effective techniques for quantitative measurement of a two- and three photon excitation efficiency spectrum of ROS sensors, We have measured the nonlinear properties of a number of new sensors: SypHer3s, HyPer7, Hypocrates, to further design multiphoton experiments with maximum brightness and dynamic response [2] in the spectral range of 700-1700 nm. In particular, a scheme for two-photon interrogation of ratiometric sensors based on a single laser oscillator has been proposed, which allows recording the dynamics of the signal arising endogenously or exogenously [3]. The optimal parameters of laser pulses for deep nonlinear optical microscopy of the mouse brain and liver have been determined. A comprehensive study of ROS biosensors in the context of two- and three-photon excitation was carried out, starting with protein solutions and ending with ratiometric imaging of sensors in models of various pathologies in living animals. Visualization with subcellular spatial resolution of the dynamics of acidity, the concentration of hydrogen peroxide and hypochlorous acid in neurons, hepatocytes and neutrophils of live mice and fish larvae with pathologies and tissue damage was demonstrated. The work was supported by the Russian Science Foundation with grants No.22-72-10044