NADPH and NADH, well known for their role in antioxidant defense and energy metabolism, are significant sources of UV-excited cellular autofluorescence, making such signals useful for cellular metabolic sensing. Previously, we applied spectral phasor analysis to autofluorescence emission during chemically induced metabolic response, showing that two-component spectral behavior, i.e., spectral change acting as a superposition of two spectra, depended on whether one or multiple metabolic pathways were affected. From this, we showed that metabolic responses primarily involving NADPH or NADH could be distinguished despite the spectral similarity of emission from these metabolic co-factors. We also showed that spectral phasors can be used to sense cellular metabolic responses even when significant spectrally similar background and absorption are present. Here, we present progress in extending such analysis to an imaging mode, thus providing spatial information during metabolic sensing. We construct a hyperspectral imaging system using a pushbroom configuration, consisting of an imaging spectrograph with high spectral resolution and a CMOS camera. Initial characterization of the system is presented here. The system can be used in both an absorption and UV-excited fluorescence mode. Assessment of the system’s ability to sense NADH emission is discussed.
Malik et al. (Sun,) studied this question.