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 e±cient signal collection deep in the brain.