We demonstrate optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic co-factors NAD(P)H and FAD to quantify metabolic changes in human immune cells from peripheral blood. This approach is attractive because it does not require cell surface labels or transfection, enabling rapid assessment of single cell metabolism. Multiphoton microscopy provides near infrared excitation of these autofluorescent molecules, thereby maximizing cell viability. Newly trained neural networks automatically segment single cells for analysis of heterogeneity within and between patients. Overall, this approach is attractive for both basic research and patient management in cancer and immunology.
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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