Impact of the emission wavelengths on in vivo multiphoton imaging of mouse brains

Tissue scattering and absorption impact the excitation and emission light in different ways for multiphoton imaging. The collected fluorescence includes both ballistic photons and scattered photons whereas multiphoton excited signal within the focal volume is mostly generated by ballistic photons. The impact of excitation wavelengths on multiphoton imaging has been extensively investigated before; however, experimental data is lacking to evaluate the impact of emission wavelengths on fluorescence attenuation in deep imaging. Here we perform three-photon imaging of mouse brain vasculature in vivo using green, red, and near-infrared emission fluorophores, and compare quantitatively the attenuation of the fluorescence signal in the mouse brain at the emission wavelengths of 520 nm, 615 nm and 711 nm. Our results show that the emission wavelengths do not significantly influence the fluorescence collection efficiency. For the green, red and near-infrared fluorophores investigated, the difference in fluorescence collection efficiency is less than a factor of 2 at imaging depths between 0.6 and 1 mm. The advantage of long wavelength dyes for multiphoton deep imaging is almost entirely due to the long excitation wavelengths.