Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

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.

Effects of Photodynamic Therapy on Tumor Metabolism and Oxygenation Revealed by Fluorescence and Phosphorescence Lifetime Imaging.

This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of photosensitizers-chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging. Oxygen content was assessed using phosphorescence lifetime macro-imaging with an oxygen-sensitive probe. For visualization of the perfused microvasculature, an optical coherence tomography-based angiography was used. It was found that PDT induces different alterations in cellular metabolism, depending on the degree of oxygen depletion. Moderate decrease in oxygen in the case of KillerRed was accompanied by an increase in the fraction of free NAD(P)H, an indicator of glycolytic switch, early after the treatment. Severe hypoxia after PDT with Photoditazine resulted from a vascular shutdown yielded in a persistent increase in protein-bound (mitochondrial) fraction of NAD(P)H. These findings improve our understanding of physiological mechanisms of PDT in cellular and vascular modes and can be useful to develop new approaches to monitoring its efficacy.

FUCCI-Red: a single-color cell cycle indicator for fluorescence lifetime imaging.

The phase of the cell cycle determines numerous aspects of cancer cell behaviour including invasiveness, ability to migrate and responsiveness to cytotoxic drugs. To non-invasively monitor progression of cell cycle in vivo, a family of genetically encoded fluorescent indicators, FUCCI (fluorescent ubiquitination-based cell cycle indicator), has been developed. Existing versions of FUCCI are based on fluorescent proteins of two or more different colors fused to cell-cycle-dependent degradation motifs. Thus, FUCCI-expressing cells emit light of different colors in different phases providing a robust way to monitor cell cycle progression by fluorescence microscopy and flow cytometry but limiting the possibility to simultaneously visualize other markers. To overcome this limitation, we developed a single-color variant of FUCCI, called FUCCI-Red, which utilizes two red fluorescent proteins with distinct fluorescence lifetimes, mCherry and mKate2. Similarly to FUCCI, these proteins carry cell cycle-dependent degradation motifs to resolve G1 and S/G2/M phases. We showed utility of FUCCI-Red by visualizing cell cycle progression of cancer cells in 2D and 3D cultures and monitoring development of tumors in vivo by confocal and fluorescence lifetime imaging microscopy (FLIM). Single-channel registration and red-shifted spectra make FUCCI-Red sensor a promising instrument for multiparameter in vivo imaging applications, which was demonstrated by simultaneous detection of cellular metabolic state using endogenous fluorescence in the blue range.

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

In vivo metabolic and SHG imaging for monitoring of tumor response to chemotherapy.

Although chemotherapy remains one of the main types of treatment for cancer, treatment failure is a frequent occurrence, emphasizing the need for new approaches to the early assessment of tumor response. The aim of this study was to search for indicators based on optical imaging of cellular metabolism and of collagen in tumors in vivo that enable evaluation of their response to chemotherapy. The study was performed on a mouse colorectal cancer model with the use of cisplatin, paclitaxel, and irinotecan. The metabolic activity of the tumor cells was assessed using fluorescence lifetime imaging of the metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H. Second harmonic generation (SHG) imaging was used to analyze the extent and properties of collagen within the tumors. We detected an early decrease in the free/bound NAD(P)H ratio in all treated tumors, indicating a shift toward a more oxidative metabolism. Monitoring of collagen showed an early increase in the amount of collagen followed by an increase in the extent of its orientation in tumors treated with cisplatin and paclitaxel, and decrease in collagen content in the case of irinotecan. Our study suggests that changes in cellular metabolism and fibrotic stroma organization precede morphological alterations and tumor size reduction, and that this indicates that NAD(P)H and collagen can be considered as intrinsic indicators of the response to treatment. This is the first time that these parameters have been investigated in tumors in vivo in the course of chemotherapy with drugs having different mechanisms of action. © 2018 International Society for Advancement of Cytometry.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis.

Fluorescence time-resolved macroimaging.

While laser scanning fluorescence lifetime imaging (FLIM) is a powerful approach for cell biology, its small field of view (typically less than 1 mm) makes it impractical for the imaging of large biological samples that is often required for biomedical applications. Here we present a system that allows performing FLIM on macroscopic samples as large as 18 mm with a lateral resolution of 15 µm. The performance of the system is verified with FLIM of endogenous metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, and genetically encoded fluorescent protein mKate2 in a mouse tumor in vivo.

Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel.

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel. It was found that, among the tested parameters (the fluorescence intensity-based redox ratio FAD/NAD(P)H, and the fluorescence lifetimes of NAD(P)H and FAD), the fluorescence lifetime of NAD(P)H is the most sensitive in tracking the drug response, and is capable of indicating heterogeneous cellular responses both in cell monolayers and in multicellular tumor spheroids. We observed that metabolic reorganization to a more oxidative state preceded the morphological manifestation of cell death and developed faster in cells that were more responsive to the drug. Our results suggest that noninvasive, label-free monitoring of the drug-induced metabolic changes by noting the NAD(P)H fluorescence lifetime is a valuable approach to characterize the responses of cancer cells to anti-cancer treatments and, therefore, to predict the effectiveness of chemotherapy.

Imaging of Intracellular pH in Tumor Spheroids Using Genetically Encoded Sensor SypHer2.

Intracellular pH (pHi) is one of the most important parameters that regulate the physiological state of cells and tissues. pHi homeostasis is crucial for normal cell functioning. Cancer cells are characterized by having a higher (neutral to slightly alkaline) pHi and lower (acidic) extracellular pH (pHe) compared to normal cells. This is referred to as a "reversed" pH gradient, and is essential in supporting their accelerated growth rate, invasion and migration, and in suppressing anti-tumor immunity, the promotion of metabolic coupling with fibroblasts and in preventing apoptosis. Moreover, abnormal pH, both pHi and pHe, contribute to drug resistance in cancers. Therefore, the development of methods for measuring pH in living tumor cells is likely to lead to better understanding of tumor biology and to open new ways for cancer treatment. Genetically encoded, fluorescent, pH-sensitive probes represent promising instruments enabling the subcellular measurement of pHi with unrivaled specificity and high accuracy. Here, we describe a protocol for pHi imaging at a microscopic level in HeLa tumor spheroids, using the genetically encoded ratiometric (dual-excitation) pHi indicator, SypHer2.

Pharmacokinetics of Chlorin e6-Cobalt Bis(Dicarbollide) Conjugate in Balb/c Mice with Engrafted Carcinoma.

The necessary precondition for efficient boron neutron capture therapy (BNCT) is control over the content of isotope 10B in the tumor and normal tissues. In the case of boron-containing porphyrins, the fluorescent part of molecule can be used for quantitative assessment of the boron content. Study Objective: We performed a study of the biodistribution of the chlorin e6-Cobalt bis(dicarbollide) conjugate in carcinoma-bearing Balb/c mice using ex vivo fluorescence imaging, and developed a mathematical model describing boron accumulation and release based on the obtained experimental data. Materials and Methods: The study was performed on Balb/c tumor-bearing mice (CT-26 tumor model). A solution of the chlorin e6-Cobalt bis(dicarbollide) conjugate (CCDC) was injected into the blood at a dose of 10 mg/kg of the animal's weight. Analysis of the fluorescence signal intensity was performed at several time points by spectrofluorimetry in blood and by laser scanning microscopy in muscle, liver, and tumor tissues. The boron content in the same samples was determined by mass spectroscopy with inductively coupled plasma. Results: Analysis of a linear approximation between the fluorescence intensity and boron content in the tissues demonstrated a satisfactory value of approximation reliability with a Spearman's rank correlation coefficient of r = 0.938, p < 0.01. The dynamics of the boron concentration change in various organs, calculated on the basis of the fluorescence intensity, enabled the development of a model describing the accumulation of the studied compound and its distribution in tissues. The obtained results reveal a high level of correspondence between the model and experimental data.

Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2.

BACKGROUND: Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2. METHODS: A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice. RESULTS: Using SypHer2, pHi was demonstrated to be 7.34±0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas. CONCLUSIONS: Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models. GENERAL SIGNIFICANCE: We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution.

Insight into redox regulation of apoptosis in cancer cells with multiparametric live-cell microscopy.

Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) was applied to monitor simultaneously apoptosis using the genetically encoded sensor of caspase-3, mKate2-DEVD-iRFP, and the autofluorescence of redox cofactors in colorectal cancer cells upon stimulation of apoptosis with staurosporine, cisplatin or hydrogen peroxide. We found that, irrespective of the apoptotic stimulus used, ROS accumulation correlated well with both the elevated pool of mitochondrial, enzyme-bound NADH and caspase-3 activation. Meanwhile, a shift in the contribution of bound NADH could develop independently of the apoptosis, and this was observed in the case of cisplatin. An increase in the proportion of bound NADPH was detected only in staurosporine-treated cells, this likely being associated with a high level of ROS production and their resulting detoxification. The results of the study favor the discovery of new therapeutic strategies based on manipulation of the cellular redox balance, which could help improve the anti-tumor activity of drugs and overcome apoptotic resistance.

Multiphoton microscopy assessment of the structure and variability changes of dermal connective tissue in vulvar lichen sclerosus: A pilot study.

In this article, we offer a novel classification of progressive changes in the connective tissue of dermis in vulvar lichen sclerosus (VLS) relying on quantitative assessment of the second harmonic generation (SHG) signal received from formalin fixed and deparaffinized tissue sections. We formulate criteria for distinguishing four degrees of VLS development: Initial-Mild-Moderate-Severe. Five quantitative characteristics (length and thickness type I Collagen fibers, Mean SHG signal intensity, Skewness and Coherence SHG signal) are used to describe the sequential degradation of connective tissue (changes in the structure, orientation, shape and density of collagen fibers) up to the formation of specific homogeneous masses. Each of the degrees has a characteristic set of quantitatively expressed features. We focus on the identification and description of early, initial changes of the dermis as the least specific. The results obtained by us and the proposed classification of the degrees of the disease can be used to objectify the dynamics of tissue changes during treatment.

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.

Monitoring membrane viscosity in differentiating stem cells using BODIPY-based molecular rotors and FLIM.

Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed "molecular rotors", in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation. In order to correlate the viscosity values with membrane lipid composition, the detailed analysis of the corresponding membrane lipid composition of differentiated cells was performed by time-of-flight secondary ion mass spectrometry. Our results directly demonstrate for the first time that differentiation of MSCs results in distinct membrane viscosities, that reflect the change in lipidome of the cells following differentiation.

Monitoring of hydrogen peroxide production under photodynamic treatment using protein sensor HyPer.

An interest to H2O2 accumulation under photodynamic treatment can be explained by its participation in intracellular signal cascades. It is important not only to detect H2O2 generation, but also to trace the dynamics of its intracellular content. In the present study the dynamics of cellular H2O2 content under photodynamic treatment was analyzed using genetically encoded reversible H2O2-sensitive sensor HyPer. Real-time detecting of H2O2 production after photodynamic treatment was performed using the protein sensor and individual features of action of different photosensitizers were revealed. Photodynamic treatment with a number of chlorin and phthalocyanine photosensitizers was found to induce secondary production of H2O2 in the cells. Three types of dynamic responses were registered: monotonous increase of H2O2 level during the entire observation time in the presence of Fotoditazin and Holosens; transient short-term accumulation in the presence of Radachlorin and Phthalosens; and relatively low-level stable increase in the presence of Photosens. The listed photosensitizers differ significantly in intracellular localization and physicochemical properties, which can determine the differences in the response of H2O2 after the photodynamic treatment. In general, it has been shown that the rapid transient H2O2 response is typical for hydrophobic compounds localized in membrane cell structures, whereas in the presence of more hydrophilic dyes a prolonged monotonous H2O2 accumulation occurs.

Photodynamic therapy of mouse tumor model using chlorin e6- polyvinyl alcohol complex.

The use of polymeric carriers to deliver hydrophobic photosensitizers has been widely discussed as a way to improve both fluorescence diagnostic and photodynamic therapy (PDT) of cancers; however, the photophysical and pharmacokinetic parameters, as well as the PDT activity, of such modifications have, until now, only been poorly investigated. The purpose of the present study was to explore the efficacy of PDT with the formulation of the photosensitizer chlorin e6 (Ce6) in combination with polyvinyl alcohol (PVA) in comparison with Ce6 alone and with the clinical drug, Photodithazine in a mouse tumor model. We also investigated the photoactivity of the Ce6-PVA in a model reaction of tryptophan oxidation, analyzed the polymer-Ce6 interaction using fluorescence spectroscopy and atomic-force microscopy, and tested the phototoxicity in vitro. Using fluorescence imaging in vivo we found that injection to mice of Ce6 in a formulation with PVA resulted in a higher tumor-to-normal ratio and greater photobleaching when compared with either the use of Ce6 alone, or with the effects of Photodithazine. Tumor growth study and histological examination of CT26 tumors revealed fast, reproducible tumor regression and more advanced necrosis after PDT with Ce6-PVA. The higher photoactivity of the Ce6-PVA complex was confirmed in a model reaction of tryptophan oxidation and in cultured cells. Therefore, encapsulation of Ce6 in PVA represents a promising strategy for further increasing the selectivity and efficacy of PDT.

Water-soluble cyclometalated platinum(ii) and iridium(iii) complexes: synthesis, tuning of the photophysical properties, and in vitro and in vivo phosphorescence lifetime imaging.

This paper presents synthesis and photophysical investigation of cyclometalated water-soluble Pt(ii) and Ir(iii) complexes containing auxiliary sulfonated diphosphine (bis(diphenylphosphino)benzene (dppb), P^P*) ligand. The complexes demonstrate considerable variations in excitation (extending up to 450 nm) and emission bands (with maxima ranging from ca. 450 to ca. 650 nm), as well as in the sensitivity of excited state lifetimes to molecular oxygen (from almost negligible to more than 4-fold increase in degassed solution). Moreover, all the complexes possess high two-photon absorption cross sections (400-500 GM for Pt complexes, and 600-700 GM for Ir complexes). Despite their negative net charge, all the complexes demonstrate good uptake by HeLa cells and low cytotoxicity within the concentration and time ranges suitable for two-photon phosphorescence lifetime (PLIM) microscopy. The most promising complex, [(ppy)2Ir(sulfo-dppb)] (Ir1*), upon incubation in HeLa cells demonstrates two-fold lifetime variations under normal and nitrogen atmosphere, correspondingly. Moreover, its in vivo evaluation in athymic nude mice bearing HeLa tumors did not reveal acute toxicity upon both intravenous and topical injections. Finally, Ir1* demonstrated statistically significant difference in lifetimes between normal tissue (muscle) and tumor in macroscopic in vivo PLIM imaging.

Multimodal label-free imaging of living dermal equivalents including dermal papilla cells.

BACKGROUND: Despite the significant progress in the development of skin equivalents (SEs), the problem of noninvasively assessing the quality of the cell components and the collagen structure of living SEs both before and after transplantation remains. Undoubted preference is given to in vivo methods of noninvasive, label-free monitoring of the state of the SEs. Optical bioimaging methods, such as cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), and fluorescence lifetime imaging microscopy (FLIM), present particular advantages for the visualization of such SEs. METHODS: In this study, we simultaneously applied several visualization techniques for skin model examination. We investigated the structure and quality of dermal equivalents containing dermal papilla (DP) cells and dermal fibroblasts (FBs) using CP OCT, MPT, and FLIM. Both the energy metabolism of the cell components and the structuring of the collagen fibrils were addressed. RESULTS: Based on the data from the fluorescence lifetimes and the contributions of protein-bound NAD(P)H, a bias toward oxidative metabolism was indicated, for the first time, in both the DP cells and FBs on day 14 of SE cultivation. The CP OCT and MPT data also indicated that both DP cells and FBs structured the collagen gel in a similar manner. CONCLUSION: In this study, multimodal label-free imaging of the structure and quality of living dermal equivalents was implemented for the first time with the use CP OCT, MPT, and FLIM of NAD(P)H. Our data suggest that the combination of different imaging techniques provides an integrated approach to data acquisition regarding the structure and quality of dermal equivalents, minimizes the potential disadvantages of using a single method, and provides an ideal information profile for clinical and research applications.

Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation.

BACKGROUND: Metabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM. METHODS: Differentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. The cells were imaged before the induction of differentiation (day 0) and on days 7, 14, and 21 of osteogenic and chondrogenic differentiation. RESULTS: Based on the data for the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound NAD(P)H, we registered a metabolic shift toward a more glycolytic status in the process of MSC differentiation. The difference was that, in osteogenic differentiation, an increase in oxidative phosphorylation preceded the shift to the glycolytic status in the process of such MSC differentiation. The fluorescence lifetime characteristics of FAD indicated the stimulation of an unknown metabolic pathway, where protein-bound FAD participates. CONCLUSIONS: In this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.