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.

Phytoplankton are extraordinarily diverse, comprising 13 phylogenetic groups, with diatoms, dinoflagellates, and haptophytes among the most prominent eukaryotes in the ocean. Development of sensor technologies for rapid taxonomic and physiological analysis of phytoplankton communities is crucial for ecological monitoring programs in the global ocean. We describe a novel, ultra-sensitive, multi-spectral fluorescence induction and relaxation instrument (a mini-FIRe) and examine its analytical capability of rapidly determining phytoplankton taxonomic groups, as well as physiological characteristics and photosynthetic rates. We collected and analyzed the database of spectral and photosynthetic properties of major taxonomic groups of phytoplankton. We revealed that the spectral shape of the functional absorption cross-section of Photosystem II (PSII), sPSII(lex), is remarkably constrained within each major phylogenetic group of eukaryotic phytoplankton, including diatoms, haptophytes, dinoflagellates, and chlorophytes. Variability in sPSII(lex) within each group was significantly smaller than the difference between groups. We also examined the classical excitation spectra of chl a fluorescence yields, Fm(lex). Our comparative analysis revealed that sPSII(lex) is a better and more specific proxy for taxonomic analysis. For instance, our developed sPSII-based algorithm correctly identified 90% of experimental data, compared to 77% identified by the Fm-based algorithm. Our results suggest that the multi-color variable fluorescence analysis offers a tool for combined physiological and taxonomic analysis, including identification of major phyla within the ‘red’ lineage of eukaryotic phytoplankton.

Non-photochemical quenching (NPQ) is a mechanism responsible for high light tolerance in photosynthetic organisms. In cyanobacteria, NPQ is realized by the interplay between light-harvesting complexes, phycobilisomes (PBs), a light sensor and effector of NPQ, the photoactive orange carotenoid protein (OCP), and the fluorescence recovery protein (FRP). Here, we introduced a biophysical model, which takes into account the whole spectrum of interactions between PBs, OCP, and FRP and describes the experimental PBs fluorescence kinetics, unraveling interaction rate constants between the components involved and their relative concentrations in the cell. We took benefit from the possibility to reconstruct the photoprotection mechanism and its parts in vitro, where most of the parameters could be varied, to develop the model and then applied it to describe the NPQ kinetics in the Synechocystis sp. PCC 6803 mutant lacking photosystems. Our analyses revealed  that while an excess of the OCP over PBs is required to obtain substantial PBs fluorescence quenching in vitro, in vivo the OCP/PBs ratio is less than unity, due to higher local concentration of PBs, which was estimated as ~10 M, compared to in vitro experiments. The analysis of PBs fluorescence recovery on the basis of the generalized model of enzymatic catalysis resulted in determination of the FRP concentration in vivo close to 10% of the OCP concentration. Finally, the possible role of the FRP oligomeric state alteration in the kinetics of PBs fluorescence was shown. This paper provides the most comprehensive model of the OCP-induced PBs fluorescence quenching to date and the results are important for better understanding of the regulatory molecular mechanisms underlying NPQ in cyanobacteria.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells’ metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.