Evaluating of metabolic activity of fibroblasts incubated on porous ceramics using fluorescence lifetime imaging and phasor approach
Tatiana B. Lepekhina 1, Viktor V. Nikolaev 1, Maxim E. Darvin 2, Hala Zuhayri 1, Mikhail S. Snegerev 1, Aleksandr S. Lozhkomoev 3, Elena I. Senkina 1,3
1 Laboratory of Laser Molecular Imaging and Machine Learning, Tomsk State University, Lenin Ave. 36, 634050 Tomsk, Russia
2 Independent Researcher, 10178 Berlin, Germany
3 Institute of Strength Physics and Materials Science of the Siberian Branch of the Russian Academy of Sciences (ISPMS SB RAS), 634021 Tomsk, Russia
Abstract
Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique in biology that analyzes the fluorescence lifetime of fluorophore molecules. FLIM has low phototoxicity, high penetration depth, enables functional and structural visualization of biological tissue and endogenous fluorophores at the cellular and subcellular levels.
The report presents a new approach for assessing the engraftment of implants in tissue based on the analysis of the viability and proliferation of 3T3 mouse fibroblasts incubated on bioinert porous materials. This approach allows to assess the metabolic processes occurring at the implant-cell interface by analyzing the FLIM decay curves on a vector graph (Phasor Plot). In this plot, the fluorescence signals are transformed into the frequency domain, where each signal is represented as a point (phasor) based on its lifetime and amplitude. The position of these points in the plot provides information about the relative contributions of different molecular states, including free and bound forms of cofactors like NAD(P)H and FAD. Graphical analysis of NAD(P)H and FAD fluorescence intensity and lifetime allowed to differentiate between their free and bound states in the cells.
The use of FLIM and the phasor approach made it possible to evaluate the ratio of free and bound forms of NAD(P)H and FAD in fibroblasts of the 3T3 line, as well as to identify changes in the parameters of the fluorescence of metabolic cofactors depending on the ceramics on which the cells were incubated.
The research was carried out with the financial support of the Ministry of Science and Higher Education of the Russian Federation (Agreement No. 075-15-2024-557 dated 04/25/2024).
Speaker
Lepekhina Tatiana
Tomsk State University
Russia
Discussion
Ask question
