The microscopic viscosity plays an essential role in cellular biophysics by controlling the rates of diffusion and bimolecular reactions within the cell interior. While several approaches have emerged that have allowed the measurement of viscosity and diffusion on a single cell level in vitro, the in vivo viscosity monitoring has not yet been realized. Here we report the use of fluorescent molecular rotors in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) to image microscopic viscosity in vivo, both on a single cell level and in connecting tissues of subcutaneous tumors in mice. We find that viscosities recorded from single tumor cells in vivo correlate well with the in vitro values from the same cancer cell line. Importantly, our new method allows both imaging and dynamic monitoring of viscosity changes in real time in live animals and thus it is particularly suitable for diagnostics and monitoring of the progress of treatments that might be accompanied by changes in microscopic viscosity.

Although cisplatin plays a central role in cancer chemotherapy, the mechanisms of cell response to this drug have been unexplored. The present study demonstrates the relationships between the intracellular pH (pHi), cell bioenergetics and the response of cervical cancer to cisplatin. pHi was measured using genetically encoded sensor SypHer2 and metabolic state was accessed by fluorescence intensities and lifetimes of endogenous cofactors NAD(P)H and FAD. Our data support the notion that cisplatin induces acidification of the cytoplasm early after the treatment. We revealedin vitrothat a capacity of cells to recover and maintain alkaline pHi after the initial acidification is the crucial factor in mediating the cellular decision to survive and proliferate at a vastly reduced rate or to undergo cell death. Additionally, we showed for the first time that pHi acidification occurs after prolonged therapyin vitroandin vivo, and this, likely, favors metabolic reorganization of cells. A metabolic shift from glycolysis towards oxidative metabolism accompanied the cisplatin-induced inhibition of cancer cell growthin vitroandin vivo. Overall, these findings contribute to an understanding of the mechanisms underlying the responsiveness of an individual cell and tumor to therapy and are valuable for developing new therapeutic strategies.

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. 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. 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. 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.

The effect of silica/gold nanoshells and titanium dioxide nanoparticles on the optical properties of skin is studied. By implementing in vivo measurements and Monte Carlo simulations, we analyze the efficiency of using these nanoparticles as contrasting agents for optical coherence tomography (OCT) imaging of skin. In vivo measurements are performed on pig skin, where nanoparticle suspension drops have been applied. The identification of skin layers is performed by comparison with corresponding histology images. Experimental results exhibit an increase in contrast of the obtained OCT images after a single nanoparticles application. Multiple applications do not lead to increase in the obtained contrast. To interpret the obtained experimental OCT images of skin and understand the mechanisms of contrasting, a set of Monte Carlo calculations is performed. The results of the simulations exhibit good qualitative agreement with the experimental images, and prove that the contrasting originates from the nanoparticles added, while the contrast of inclusion originates from the absence of nanoparticles within it and their presence in the surrounding area.