Phasor-based approach for resolving multicomponent fluorescence signals in frequency-domain FLIM
Ilya A. Goryunov, Daniil Y. Popov, Andrey Y. Vinokurov, Elena V. Potapova, Viktor V. Dremin; Orel State University named after I.S. Turgenev, Orel, Russia
Abstract
FLIM (fluorescence lifetime imaging microscopy) technology is one of the main optical tools for assessing the metabolic properties of biological systems. Fluorescence lifetime is a unique characteristic that depends on a molecule’s microenvironment. This makes it possible to describe both intramolecular processes and the influence of microenvironmental factors on a fluorophore. When studying multicomponent biological systems, the generation of autofluorescence signals from the main metabolic cofactors is a complex process. In general, a situation may arise in which the number of dominant fluorescent components cannot be known in advance. At the same time, standard approaches to interpreting changes in lifetime parameters may describe non-selective changes occurring in the system itself, without allowing the contribution of an individual component to be determined.
In this work, we propose a new approach based on mathematical modeling of a multicomponent fluorescent system represented in the frequency domain. Modeling allows us to establish the main patterns of change in the resulting signal when the contributions and lifetimes of individual fluorophores and their groups in the mixture are varied. To determine the contribution of the target fluorophore, phasor space and the patterns obtained by varying the model's temporal parameters are used. To test the proposed approach experimentally, a frequency-domain FLIM system with a modulation frequency of 40 MHz was used. Flavins, which act as a multicomponent source of autofluorescence, were the object of the study. To differentiate fluorescent components, isolated mitochondria from the livers of Wistar rats were examined.
The results demonstrated the performance of the presented model of a multicomponent system, which was verified using a calibrated frequency-domain FLIM system. Studies of isolated mitochondria demonstrated the possibility of differentiating the contributions of the flavin cofactors of electron transport chain complex I (FMN) and complex II (FAD). Phasor analysis allowed us to estimate the apparent fluorescence lifetime of each cofactor associated with these complexes within the complex multicomponent system.
Speaker
Ilya Goryunov
Orel State University named after I.S. Turgenev, Orel, Russia
Orel
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