MitoRACE: evaluating mitochondrial function in vivo and in single cells with subcellular resolution using multiphoton NADH autofluorescence

Key points We developed a novel metabolic imaging approach that provides direct measures of the rate of mitochondrial energy conversion with single‐cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). Measures of mitochondrial NADH flux by mitoRACE are sensitive to physiological and pharmacological perturbations in vivo. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells with potential for broad applications in the study of energy metabolism. Mitochondria play a critical role in numerous cell types and diseases, and structure and function of mitochondria can vary greatly among cells or within different regions of the same cell. However, there are currently limited methodologies that provide direct assessments of mitochondrial function in vivo, and contemporary measures of mitochondrial energy conversion lack the spatial resolution necessary to address cellular and subcellular heterogeneity. Here, we describe a novel metabolic imaging approach that provides direct measures of mitochondrial energy conversion with single‐cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). MitoRACE measures the rate of NADH flux through the steady‐state mitochondrial NADH pool by rapidly inhibiting mitochondrial energetic flux, resulting in an immediate, linear increase in NADH fluorescence proportional to the steady‐state NADH flux rate, thereby providing a direct measure of mitochondrial NADH flux. The experiments presented here demonstrate the sensitivity of this technique to detect physiological and pharmacological changes in mitochondrial flux within tissues of living animals and reveal the unique capability of this technique to evaluate mitochondrial function with single‐cell and subcellular resolution in different cell types in vivo and in cell culture. Furthermore, we highlight the potential applications of mitoRACE by showing that within single neurons, mitochondria in neurites have higher energetic flux rates than mitochondria in the cell body. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells, with potential for broad applications in the study of energy metabolism. Key points We developed a novel metabolic imaging appr