Time-Resolved Fluorescence Polarization Spectroscopy Reveals the Origin of the Spectroscopic Properties of Natural Heterogeneous Molecular Systems
Egor V. Kvasov1, Ioanna A. Gorbunova2, Yaroslav M. Beltukov3, Maxim E. Sasin3, Boris P. Yakimov1, Irina V. Perminova4, Peter S. Timashev2, 5, Evgeny A. Shirshin1,2; 1Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia; 2Institute for regenerative medicine, Sechenov First Moscow State Medical University, Moscow, Russia; 3Ioffe Institute, Saint-Petersburg, Russia; 4Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia; 5Department of Biological Chemistry, Sechenov First Moscow State Medical University, Moscow, Russia
Abstract
Heterogeneous molecular systems play an important role in photoaging, oxidative stress, and the accumulation of metabolic products in living cells and tissues. Their optical spectra exhibit several characteristic features, including broad, structureless absorption extending from the ultraviolet to the visible range, broad fluorescence bands with large full widths at half-maximum, a strong dependence of fluorescence spectra on excitation wavelength, and a gradual red shift of fluorescence emission with increasing excitation wavelength. Establishing the physical origin of these spectral properties remains challenging because individual chromophores cannot be readily isolated using conventional spectroscopic methods. In this work, we demonstrate that the analysis of fluorescence anisotropy and fluorescence depolarization time of natural and model heterogeneous systems, including humic substances, synthetic melanin, and oxidized tryptophan, provides evidence that their optical properties arise from a superposition of non-interacting fluorescent species. We found that the fundamental fluorescence anisotropy and nanosecond depolarization times strongly depended on excitation wavelength, fluorescence detection window, and the molecular size of humic substance fractions. An important finding is that the excitation-wavelength dependence of fluorescence anisotropy can be described by a superposition of fluorophores exhibiting a pronounced sigmoidal increase in anisotropy with excitation wavelength, a behavior characteristic of many aromatic heterocyclic molecules. To test this interpretation, fluorescence anisotropy was also measured for individual organic dyes. Comparison of the results obtained for these dyes and humic substances allows polarization-response features arising from the intrinsic photophysical properties of organic molecules to be distinguished from those associated with the molecular heterogeneity of natural organic systems.
Speaker
Egor V. Kvasov
Faculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia
Russia
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