Abstract
Multiphoton fluorescence microscopy enables deep in vivo imaging by using long excitation wavelengths to increase the penetration depth of ballistic photons and nonlinear excitation to suppress the out-of-focus fluorescence. However, the imaging depth of multiphoton microscopy is limited by tissue scattering and absorption. This fundamental depth limit for two-photon microscopy has been studied theoretically and experimentally. Long wavelength three-photon fluorescence microscopy was developed to image beyond the depth limit of two-photon microscopy and has achieved unprecedented in vivo imaging depth. Here we extend the theoretical framework for characterizing the depth limit of two-photon microscopy to three-photon microscopy. We further verify the theoretical predictions with experimental results from tissue phantoms. We demonstrate experimentally that high spatial resolution diffraction-limited imaging at a depth of 10 scattering mean free paths, which is nearly twice the transport mean free path, is possible with multiphoton microscopy. Our results indicate that the depth limit of three-photon microscopy is significantly beyond what has been achieved in biological tissues so far, and further technological development is required to reach the full potential of three-photon microscopy.