Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy

Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy

Blog Article

Three-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain.The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored.In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue.We show that this excitation regime provides a Sunglasses more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering.We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with Drop-In Sinks live tissue imaging.

Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging.Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs.