Abstract
Multiphoton microscopy (MPM) is a powerful imaging technology for brain research. The imaging depth in MPM is partly determined by emission wavelength of fluorescent labels. It has been demonstrated that a longer emission wavelength is favorable for signal detection as imaging depth increases. However, there has been no comparison with near-infrared (NIR) emission. In order to quantitatively analyze the effect of emission wavelength on 3-photon imaging of mouse brains in vivo, we utilize the same excitation wavelength to excite a single fluorescent dye and simultaneously collect NIR and orange-red emission fluorescence at 828[Formula: see text]nm and 620[Formula: see text]nm, respectively. Both experimental and simulation results show that as the imaging depth increases, NIR emission decays less than orange-red fluorescent emission. These results show that it is preferable to shift the emission wavelength to NIR to enable more efficient signal collection deep in the brain.
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