Refractive index and pulse broadening characterization using oil immersion and its influence on three-photon...

Three-photon microscopy excited at the 1700-nm window enables deep-tissue penetration. However, the refractive indices of commonly used immersion oils, and the resultant pulse broadening are not known, preventing imaging optimization. Here, we demonstrate detailed characterization of the refractive index, pulse broadening and distortion for excitation pulses at this window for commonly used immersion oils. On the physical side, we uncover that absorption, rather than material dispersion, is the main cause of pulse broadening and distortion. On the application side, comparative three-photon imaging results indicate that 1600-nm excitation yields 5 times higher three-photon signal than 1690-nm excitation.