Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing

Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development. Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues 1, 2. Pulsed near infrared excitation light allows in-depth imaging based on contrasts such as endog-enous fluorescence 2 , second harmonic generation (SHG) 3 and third harmonic generation (THG) 4. Endogenous fluorescence in living tissues arises from several intrinsic biomarkers that play important roles in physiological processes 2. The primary intracellular sources are NAD(P)H and FAD, the two major cofactors of redox reactions in the cell and central regulators of energy production and metabolism 5, 6. Their fluorescence reports on the metabolic activity of cells allowing tissue physiology and processes such as stem cell differentiation, cancer development and neurodegenerative diseases to being non-invasively monitored 7–12. The fluorescence lifetimes of NADH and FAD are different upon binding to the protein during the electron transport chain. FLIM provides sensitive measurements of the free and protein-bound NAD(P)H ratio and of the redox states (NADH/NAD +) of cells, and can be used to distinguish glycolytic and oxidative phosphorylation metabolic states 13–17. Monitoring lifetime of free and protein-bound FAD has also been exploited to quantify redox ratio FAD/FADH 2 , and used as a biomarker of precancerous epithelial cells 12. It is well established that retinoids play a crucial role in stem cell differentiation and embryo development 18, 19 and their concentration and gradients have been detected in vivo during zebrafish development 9, 20. Other intrinsic fluorophores such as porphyrin, collagen, elastin, keratin,