Simultaneous Two-Photon Fluorescence Microscopy of NADH and FAD Using Pixel-to-Pixel Wavelength-Switching

Two-photon fluorescence (TPF) microscopy of intrinsic fluorophores provides physiological and pathological information from biological tissues. Reduced nicotinamide adenine dinucleotide (NADH) flavin (FAD) are two endogenous fluorescent coenzymes existing on the intracellular scale. Autofluorescence images NADH FAD have been applied to noninvasively record changes during metabolism, according their distributions concentrations. However, widely used sequential (non-simultaneous) excitation scheme results in artifacts caused by sample motion or laser power fluctuation. The single-wavelength illumination suffers low efficiency spectral bleed-through. In this paper, we demonstrate a new imaging system simultaneously capturing autofluorescence FAD, with high negligible Two temporally multiplexed spatially overlapped beams were achieved fast-switching light paths based an electro-optic modulator. switching centered at 750 860 nm, enabling independent excitations FAD. acquired wavelength ranges 415–455 nm 500–550 respectively. modulator was synchronized pixel clock microscope, achieving pixel-to-pixel wavelength-switching. capability demonstrated performing TPF freshly excised mouse colon microenvironment wall depicted colonocytes, goblet cells, crypts Lieberkühn, relative concentrations estimated. experimental show that can effectively perform simultaneous is considered promising tool for investigations into metabolism-associated processes diseases.