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

Abstract

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 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 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.

Restoration of mitochondria axonal transport by adaptor Disc1 supplementation prevents neurodegeneration and rescues visual function

“…Due to its deeper anatomical location, RGC axons in the optic nerve head are inaccessible using two-photon live imaging. Given that the optic nerve head undergoes early pathological changes in glaucoma, future efforts are warranted to monitor mitochondrial movement by in vivo optical microendoscopy (Barretto and Schnitzer, 2012) or three-photon excitation intravital microscopy (Bakker et al, 2022). Mitochondrial volume assessment in single axons was performed in double transgenic mice (Thy1-CFP.MitoS::Thy1-YFP), and although we did not detect noticeable differences between single and double transgenic mice, we cannot rule out potential limitations inherent to the use of the same promoter.…”

Second Near‐Infrared (NIR‐II) Window for Imaging‐Navigated Modulation of Brain Structure and Function

“…[ 56 ] Past research demonstrated the feasibility of four‐photon fluorescence microscopy (4PM) in biological tissues based on quantum perturbation theory and measurements. [ 53,57 ] Meanwhile, the longer excitation wavelength in 4PM may cause deeper penetration and better resolution in brain tissue. However, the advantage of 4PM with excitation of NIR‐II light in terms of deep brain imaging is needed further investigation, due to the exist fluorophores with small four‐photon adsorption cross‐section.…”

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy

“…The pump and OPA wavelengths were centered at 1030 and 1320 nm respectively, resulting in TSFG and THG wavelengths at λ TSFG 1 ¼ 370 nm, λ TSFG 2 ¼ 402 nm and λ THG 2 ¼ 440 nm. We remind that OPA-based excitation at 1 MHz is expected to provide an 80 2 increase in THG efficiency compared to 80 MHz OPO excitation with similar pulse durations and average power, enabling to work at larger depths by progressively increasing the power delivered to the tissue surface 59 . One difference with the transmission geometry used in the previous section is that the epidetected signals correspond to THG and TSFG light that is mostly forward-generated and then scattered back towards the objective by tissue structures underneath the imaging plane.…”