Fluorescence lifetime imaging measures the time a fluorophore remains in the excited state before returning to the ground state. Fluorescence lifetime measurements provide environmental information about fluorophores. For the endogenous metabolic co-enzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), the fluorescence lifetime is different for free and protein-bound molecules; thus, lifetime imaging provides metabolic information about cells in a label-free manner. However, fluorescence lifetime analysis via traditional decay fitting methods is time-consuming and requires expertise. Furthermore, direct relationships between lifetime metrics and cell metabolism or functions are difficult to define. Recent advances in the combination of machine learning and fluorescence lifetime imaging allow accelerated image analysis and cell phenotype identification. Guidelines for ensuring rigor and transference of machine learning models will be discussed in the context of the development and testing of machine learning models that identify metabolism pathway use of individual cells from autofluorescence lifetime images.
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