Intravital Deep-Tumor Single-Beam 2-, 3- and 4-Photon Microscopy

Abstract

Three-photon excitation has recently been introduced to perform intravital microscopy in deep, previously inaccessible layers of the brain. The applicability of deep-tissue three-photon excitation in more heterogeneously structured, dense tissue types remains, however, unclear. Here we show that in tumors and bone, high-pulse-energy low-duty-cycle infrared excitation near 1300 and 1700 nm enables two-up to fourfold increased tissue penetration compared to conventional 2-photon excitation. Using a single laser line, simultaneous 2-, 3- and 4-photon processes are effectively induced, enabling the simultaneous detection of blue to far-red fluorescence together with second and third harmonic generation. This enables subcellular resolution at power densities in the focus that are not phototoxic to live cells and without color aberration. Thus, infrared high-pulse-energy low-duty-cycle excitation advances deep intravital microscopy in strongly scattering tissue and, in a single scan, delivers rich multi-parameter datasets from cells and complex organ structures.