An Efficient Multicolor Two-Photon Imaging of Endogenous Fluorophores in Living Tissues by Wavelength Mixing

Abstract

Two-photon imaging of endogenous fluorescence can provide important physiological and metabolic information from intact tissues in a label-free and non-invasive way. However, imaging of multiple intrinsic fluorophores, such as NADH, FAD, retinoids and porphyrins in living systems is generally hampered by sequential multi-wavelength excitation resulting in long acquisition times and motion artifacts. We report an efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 700-1040nm range, using wavelength mixing. By using two synchronized pulse trains at two different wavelengths, an additional “virtual” two-photon excitation wavelength is generated, and simultaneous excitation of blue, green and red endogenous fluorophores is achieved. This method permits fast and reliable simultaneous imaging of the metabolic coenzymes NADH and FAD to being implemented, overcoming the difficulties associated with their difference in absorption spectra and disparity in concentration. We achieve efficient ratiometric redox imaging and simultaneous efficient two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. Lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states were measured in both reconstructed human skins and live C. elegans worms. Finally, we perform hyperspectral imaging of endogenous fluorophores during early zebrafish development.