Orange carotenoid protein (OCP) is the photoactive protein that is responsible for high light tolerance in cyanobacteria. We studied the kinetics of the OCP photocycle by monitoring changes in its absorption spectrum, intrinsic fluorescence, and fluorescence of the Nile red dye bound to OCP. It was demonstrated that all of these three methods provide the same kinetic parameters of the photocycle, namely, the kinetics of OCP relaxation in darkness was biexponential with a ratio of two components equal to 2:1 independently of temperature. Whereas the changes of the absorption spectrum of OCP characterize the geometry and environment of its chromophore, the intrinsic fluorescence of OCP reveals changes in its tertiary structure, and the fluorescence properties of Nile red indicate the exposure of hydrophobic surface areas of OCP to the solvent following the photocycle. The results of molecular-dynamics studies indicated the presence of two metastable conformations of 3'-hydroxyechinenone, which is consistent with characteristic changes in the Raman spectra. We conclude that rotation of the β-ionylidene ring in the C-terminal domain of OCP could be one of the first conformational rearrangements that occur during photoactivation. The obtained results suggest that the photoactivated form of OCP represents a molten globule-like state that is characterized by increased mobility of tertiary structure elements and solvent accessibility.

Blood cell analysis is one of the standard clinical tests. Despite the widespread use of exogenous markers for blood cell quantification, label-free optical methods are still of high demand due to their possibility for in vivo application and signal specific to the biochemical state of the cell provided by native fluorophores. Here we report the results of blood cell characterization using label-free fluorescence imaging techniques and flow-cytometry. Autofluorescence parameters of different cell types -white blood cells, red blood cells, erythrophagocytic cells -are assessed and analyzed in terms of molecular heterogeneity and possibilities of differentiation between different cell types in vitro and in vivo.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Humification is a ubiquitous natural process of biomass degradation that creates multicomponent systems of nonliving organic matter, including dissolved organic matter (DOM) and humic substances (HS) in water environments, soils, and organic rocks. Despite significant differences in molecular composition, the optical properties of DOM and HS are remarkably similar, and the reason for this remains largely unknown. Here, we employed fluorescence spectroscopy with (sub)­picosecond resolution to elucidate the role of electronic interactions within DOM and HS. We revealed an ultrafast decay component with a characteristic decay lifetime of 0.5–1.5 ps and spectral diffusion originating from excitation energy transfer (EET) in the system. The rate of EET was positively correlated to the fraction of aromatic species and tightness of aromatic species packing. Diminishing the number of EET donor–acceptor pairs by reduction with NaBH4 (decrease of the acceptor number), decrease of pH (decrease of the electron-donating ability), or decrease of the average particle size by filtration (less donor–acceptor pairs within a particle) resulted in a lower impact of the ultrafast component on fluorescence decay. Our results uncover the role of electronic coupling among fluorophores in the formation of DOM and HS optical properties and provide a framework for studying photophysical processes in heterogeneous systems of natural fluorophores.

Most cyanobacteria utilize a water-soluble Orange Carotenoid Protein (OCP) to protect their light-harvesting complexes from photodamage. The Fluorescence Recovery Protein (FRP) is used to restore photosynthetic activity by inactivating OCP via dynamic OCP–FRP interactions, a multistage process that remains underexplored. In this work, applying time-resolved spectroscopy, we demonstrate that the interaction of FRP with the photoactivated OCP begins early in the photocycle. Interacting with the compact OCP state, FRP completely prevents the possibility of OCP domain separation and formation of the signaling state capable of interacting with the antenna. The structural element that prevents FRP binding and formation of the complex is the short α-helix at the beginning of the N-terminal domain of OCP, which masks the primary site in the C-terminal domain of OCP. We determined the rate of opening of this site and show that it remains exposed long after the relaxation of the red OCP states. Observations of the OCP transitions on the ms time scale revealed that the relaxation of the orange photocycle intermediates is accompanied by an increase in the interaction of the carotenoid keto group with the hydrogen bond donor tyrosine-201. Our data refine the current model of photoinduced OCP transitions and the interaction of its intermediates with FRP.