Probing cellular metabolism and embryo development by fluorescence lifetime microscopy of endogenous biomarkers

Here we present recent advances in label-free metabolic imaging of living tissues by two-Photon Fluorescence Lifetime Microscopy (2P-FLIM) of endogenous biomarkers. We implement simultaneous two-photon excitation of NAD(P)H and FAD by wavelength mixing to acquire 2P-FLIM data of the two biomarkers at the same time and perform efficient multiparametric metabolic imaging in dynamic biological system. We demonstrate how 2P-FLIM of the two metabolic coenzymes reveals the richness and complexity of several metabolic processes in intact tissues with minimal phototoxicity and can be implemented non-invasively for longitudinal studies in vitro and in vivo such as stem cell differentiation and embryo development.