Quantitative oblique back-illumination microscopy (qOBM) is a label-free imaging technique that enables tomographic phase imaging of thick scattering samples with epi-illumination. Here, we propose the use of two forms of functional imaging with qOBM to study tissue and cell cultures. In doing so, we obtain the spatiotemporal and quantitative functional information associated with the phase values extrapolated from qOBM imaging. We have applied this process to study the efficacy of individual immune T cells to kill glioblastoma spheroid cultures in 3D spheroids. Data show that we can effectively distinguish between cell phenotypes and characterize the dynamic motion of these cells in 3D cultures. This work offers a distinct advantage in tracking 3D cellular dynamics in thick tissue as many function imaging modalities are limited to 2D samples. Further, this technology can be expanded to analyze a wide variety of cellular and subcellular dynamics non-invasively in thick tissue.
Human pluripotent stem cell (hPSC)-derived retinal cell culture holds great promise for treatment of retinal diseases that cause blindness. However, high-throughput non-invasive functional assays are needed for rapid optimization and quality control of these stem cell culture systems. Here we use simultaneous two- and three-photon excited fluorescence lifetime imaging microscopy (FLIM) to characterize the autofluorescent signature of light response in photoreceptor cells that originates from the fluorescent vitamin A compounds (i.e., retinoids) of the visual cycle. This multiphoton microscopy technique resolves the dynamics of visual pigment photobleaching and recycling following light exposure, including conversion of different retinoids to one another.
Current methods to assess immune cell function use labels that are limiting for time-course studies of immune cell behavior in tumors. Here, we use multiphoton imaging of NAD(P)H and FAD, co-enzymes of metabolism, in T cells and macrophages within the tumor microenvironment. T cells alter their metabolism in response to tumor-like pH, glucose, and lactic acid levels, while macrophages alter their metabolism during tumor-stimulated migration. These results indicate that multiphoton autofluorescence imaging is a powerful label-free method to monitor immune cell metabolism within single cells in the tumor microenvironment.
Antitumor activity of T cells often determines treatment efficacy for immunotherapy and radiotherapy. Due to the immunosuppressive tumor microenvironment, tumor-infiltrating T cells often exhibit a hypofunctional exhausted phenotype. Current methods to evaluate T cell exhaustion use flow cytometry or cytokine production measurements that are either destructive to the sample or cannot capture single-cell heterogeneity, respectively. Here, we used fluorescence lifetime imaging of the metabolic co-enzyme NAD(P)H to evaluate T cell metabolism during exhaustion. Exhausted T cells have significantly different NAD(P)H lifetimes compared to functional T cells. This study demonstrates a label-free method to monitor T cell exhaustion in tumors.
Current methods to assess T cell function use labels that prevent non-destructive quality control of T cell infusions. Here, we use autofluorescence imaging of NAD(P)H and FAD, co-enzymes of metabolism, in quiescent and activated T cells for label-free, non-destructive determination of T cell activation state and subtype. Logistic regression models achieved 97-99% accuracy for classification of T cell activation, and random forest models of achieved >97% accuracy for four-group classification of quiescent and activated CD3+CD8+ and CD3+CD4+ T cells. These results indicate that NAD(P)H and FAD imaging is a powerful method for label-free, non-destructive quality control of T cell infusions.
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