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ALT'23 The 30th International Conference on Advanced Laser Technologies
B-O-11
Time-resolved fluorescence imaging of lipofuscin, incorporated in vitro into retinal pigment epithelial cells: Effects of photooxidation and protein-mediated antioxidant delivery
A.N. Semenov1, E. G. Maksimov1, A. M. Moysenovich1, M. A. Yakovleva2, G. V. Tsoraev1, E. A.
Shirshin134, N. N. Sluchanko5, T. B. Feldman12, A. B. Rubin1, M. P. Kirpichnikov1, Mikhail A. Ostrovsky12
1- M.V. Lomonosov Moscow State University 2- Emanuel Institute of Biochemical Physics, Russian Academy of Sciences
3- World-Class Research Center "Digital Biodesign and Personalized Healthcare", Sechenov First Moscow State
Medical University 4- Institute of Spectroscopy, Russian Academy of Sciences 5- Federal Research Center of Biotechnology, Russian Academy of Sciences
semenov@physics. msu. ru
Lipofuscin of retinal pigment epithelium (RPE) cells is a complex of chromophores accumulating as intracellular granules during the cell's lifespan. Being exposed to light, lipofuscin becomes a source of oxidative stress. It significantly complicates the course of various age-related eye diseases and without proper control may lead to the irreversible loss of the vision. Modern clinical routine utilizes fundus autofluorescence (FAF) imaging to assess the lipofuscin concentration in RPE. However, since FAF is based on the measurements of the intensity of lipofuscin fluorescence, very often this method does not allow to diagnose the pathology on the early stage. As a possibility to increase the sensitivity, the usage of the time-resolved measurements of lipofuscin fluorescence is being proposed. The aim of the work was to assess the lifetime of autofluorescence of lipofuscin, incorporated in vitro into RPE cells, with and without photooxidation in order to estimate lifetime as a diagnostic parameter. Additionally, we measured how administration of carotenoid antioxidant zeaxanthin via protein-mediated mechanisms affects lipofuscin fluorescence lifetime.
Lipofuscin granules (LG) were incorporated into RPE cell line culture ARPE-19 obtained from Koltzov Institute of Developmental Biology of RAS (Moscow, Russia). Fluorescence lifetime imaging (FLIM) was performed in the time-correlated single photon-counting (TCSPC) mode using the confocal system installed on the Eclipse Ti2 (Nikon, Tokyo, Japan) microscope. Excitation was performed by a 473 nm picosecond laser (30 ps impulse duration, 50 MHz repetition rate) synchronized with detector HMP-100-40 via board SPC-150 (Becker&Hickl, Germany). The detection was performed using a cutting-band filter at 530 nm with 40 nm band width (Thorlabs, USA). The photooxidation of cells enriched with LG was performed in CO2-incubator by 18h-illumination with a custom LED array (light intensity 0.3 mW/cm2). Zeaxanthin was administered into ARPE-19 cells in complex with BombyxMori Carotenoid-Binding Protein (BmCBP-ZEA) at 200 nM concentration (calculated by the protein). The description of BmCBP production, as well as its abilities to bind various carotenoids, and demonstration of its antioxidant properties, are available in our previous work [1].
The analysis of the FLIM data of ARPE-19 cells, enriched with LG, revealed that autofluorescence of intact lipofuscin was characterized with mean lifetime Tmean = 275±45 ps. Irradiation of cells led to the massive photooxidation, and the significant increase in lipofuscin fluorescence lifetime (375±37 ps) was detected. Supplement of cells with BmCBP-ZEA resulted in lower values of the lipofuscin fluorescence mean lifetime after illumination (310±23 ps), demonstrating the suppression of the photooxidation due to the antioxidation effect. For more details please refer to our work [2].
Basing on the obtained results, we conclude that time-resolved diagnostics of lipofuscin autofluorescence is sensitive to the photooxidation processes, as well as to the effects of carotenoid antioxidants. Thus, it can be proposed as a part of fluorescence imaging ophthalmoscopy technique.
The present work was supported by Russian Scientific Foundation grant № 22-25-00183.
[1] N. Sluchanko, Y. Slonimskiy, E. Maksimov et al., Silkworm carotenoprotein as an efficient carotenoid extractor, solubilizer and transporter, International Journal of Biological Macromolecules, vol. 223, pp. 1381-1393 (2022).
[2] A. Semenov, E. Maksimov, N. Sluchanko et al., Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in
Retinal Pigment Epithelial Cells, IJMS, vol. 12 (413), pp. 1-19, (2023).
