Comparison of the emission wavelengths by a single fluorescent dye on in vivo 3-photon imaging of mouse brains

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 nm and 620 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.