Multiphoton microscopy of reduced Nicotinamide Adenine Dinucleotide(phosphate) (NAD(P)H) is an excellent tool for exploring metabolism at the single-cell level. We present the application of fluorescence anisotropy in multiphoton autofluorescence imaging. We explore time-resolved methods that can be used for metabolic interpretations: 1) the time-resolved anisotropy, 2) time-gated anisotropy and 3) steady-state anisotropy methods. These imaging schemes are achieved using a time-correlated single-photon counting microscope, allowing simultaneous pixel-wise registration of fluorescence intensity, lifetime, and anisotropy. This tool is well-suited to identify the enzyme binding states and offer new biophysical definitions for NAD(P)H imaging. We demonstrate this hyperdimensional imaging modality under an open-sourced imaging platform.

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