To develop new methods for the diagnosis and treatment of such a widespread disease as steatosis, there is still a lack of fundamental biological knowledge about various aspects of the functioning of the liver tissue at the cellular level. In our work, we assessed the metabolic state of hepatocytes, as well as the collagen content in the liver tissue with induced steatosis using the modern label-free minimally invasive methods of multiphoton laser scanning microscopy with TPEF mode (Two-Photon Excited Fluorescence) and SHG mode (Second Harmonic Generation), equipped with FLIM (Fluorescence-Lifetime Imaging Microscopy). Using multiphoton microscopy, it was shown that during the development of steatosis, it is possible to identify areas with a reduced NAD(P)H autofluorescence signal in damaged hepatocytes. Using SHG we showed a gradual accumulation of collagen in the liver tissue with induced steatosis, however, extensive areas of fibrosis were not detected even at the advanced stages of the pathology. Using FLIM, we studied the specific features of the energy metabolism of hepatocytes based on data on the lifetimes of various forms of NAD(P)H and their relative contributions. It has been revealed that there is a gradual decrease in the intensity of oxidative phosphorylation, accompanied by the rise in the intensity of lipogenesis in the liver tissue with induced steatosis. Such results are consistent with the data of histological analysis. The results obtained in this work can be useful for developing new criteria for express intraoperative assessment of liver pathology at the cellular level in the clinic.
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