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Possibilities of two-photon microscopy for analysis of fluorescent properties of elastin fibers of rats in vivo

Anastasia I. Knyazkova1,2, Aliсe A. Samarinova 2, Viktor V. Nikolaev 2, Alexey V. Borisov 2, Yury V. Kistenev 2,3
1V.E. Zuev Institute of Atmospheric Optics SB RAS, Tomsk 634055, Russia
2Tomsk State University, Tomsk 634050, Russia
3Siberian State Medical University, Tomsk 634050, Russia


Two-photon microscopy is one of the variants of laser scanning microscopy, in which fluorochromes are excited by laser radiation of the infrared or long-wavelength visible range with high intensity, while the emission occurs at a wavelength half the wavelength of the incident radiation.Due to the fact that the emission spectra of many fluorescent molecules are close enough, the analysis of the obtained images becomes difficult in the case of their joint study. The fluorescence lifetime estimation method (FLIM) makes it possible to distinguish between individual fluorophores over an interval corresponding to the fluorescence lifetime. This method consists in analyzing the fluorescence decay curve for different molecules. FLIM-fluorescence of tissues also provides a high contrast with fluorescent images obtained under the action of endogenous fluorophores in biological tissues, allowing to study the interaction of proteins and their conformational transformations.Two-photon fluorescence microscopy in combination with FLIM allows monitoring the content of, for example, molecules such as tryptophan, tyrosine, collagen and elastin in tissue. The possibility of detecting photons in very short time intervals makes it possible to assess the metabolic status of cells by analyzing the temporal dynamics of fluorescence.
This paper presents the results of in vivo visualization of elastin fibers of the papillary layer of rat skin obtained using two-photon microscopy. It was shown that, when approximating the fluorescence decay curve by a two-exponential model, the obtained parameters for rat dermis elastin and human elastin fibers have similar distributions.
This presentation is devoted to the use of the method of two-photon microscopy to study the fluorescent properties of elastic fibers of the dermis of rat skin. The emphasis is on the analysis of fluorescence decay curves. In this work, the following fluorescent characteristics of rat dermis elastin were determined: the average fluorescence lifetime, the fast and slow component lifetimes, and the ratio of the amplitudes of the slow and fast components. The above parameters were compared with the parameters of human dermal elastin fibers taken from the literature. The obtained distributions make it possible to assess the possibility of comparing the fluorescent characteristics of human and rat elastin, and then generalize to the case of pathologies, which will allow us to proceed to the study of pathologies associated with changes in the fluorescence parameters of human skin in small animal models.
This work was performed within the frame partial financial support from the Russian Foundation for Basic Research (grant №20-32-90098) and carried out with partial support of a grant under the Decree of the Government of the Russian Federation No. 220 of 09 April 2010 (Agreement No. 075-15-2021-615 of 04 June 2021).


Samarinova Alice Andreevna
Laboratory of Laser Molecular Imaging and Machine Learning, Tomsk State University


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