Abstract
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.
Highlights
Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues[1, 2]
While several advanced approaches have been developed to improve identification and quantification of endogenous fluorophores based on fluorescence lifetime imaging[9], spectrally resolved detection[23, 25], and spectrally resolved FLIM26–28, multiphoton microscopy of endogenous fluorophores and its applications to live imaging are still limited by acquisition speed and challenging multicolor excitation
Imaging of multiple endogenous fluorophores is usually performed by sequential excitation at different wavelengths using a tunable laser, leading to imaging rates decreasing with the number of excitation wavelengths, and difficulties in ensuring pixel-level registration between the channels in the case of a dynamic sample
Summary
Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues[1, 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[7,8,9,10,11,12]. Strategies conceived so far to simultaneously excite NADH and FAD with one single wavelength[32] can compensate the difference in absorption spectra and concentrations with the payoff of low excitation efficiency To overcome these limitations, a mixed-wavelength excitation[33] was implemented to achieve multicolor two-photon imaging of endogenous fluorophores in living tissues. A simultaneous imaging of several endogenous fluorophores and SHG signals in living zebrafish embryos is demonstrated
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