Three-photon microscopy (3PM) has emerged as a versatile intravital imaging technology, enabling analysis of cell dynamics and interactions with the tissue microenvironment beyond the capabilities of two-photon microscopy (2PM) by providing access to deeper layers in highly scattering tissues. Due to the low probability of three-photon processes, higher photon flux densities are required compared to 2PM when using similar moderate average laser powers to prevent tissue photodamage. Consequently, pulse broadening compensation is imperative, in addition to optimal beam focusing. However, the phenomena contributing to pulse broadening in biological tissues are largely understudied. We measured the pulse broadening of a 1650 nm, 65 fs pulsed laser beam in various musculoskeletal and lymphoid tissues and its dependence on dispersion compensation by zinc selenide (ZnSe). By employing a model function based on the nonlinear Schrödinger equation to approximate the experimental data, we were able to determine the tissue-specific contributions of second- and third-order dispersion to pulse broadening. This analysis revealed additional dispersion-independent contributions that are dependent on the laser power. Furthermore, our study showed that the ZnSe-based dispersion compensation depends on the tissue depth, as demonstrated by the analysis of the third-harmonic generation signal levels. Our results suggest that dispersion compensation adapted to tissue type and imaging depth can significantly improve 3PM performance, especially in challenging musculoskeletal and lymphoid organs.
