Session Index

We have achieved video-rate three-photon fluorescence microscopy based on a 1320-nm femtosecond source, driven by a single 24-MHz Cr:forsterite oscillator and fiber-optic nonlinear conversion. This demonstration provides a solution for robust a source system for deep-tissue GFP imaging. We optimized the laser oscillator to deliver 40-nJ output pulse energy at around 1260 nm, and the matching of the output wavelength and GFP excitation can be realized by precisely managing the interplay between self-phase modulation and fiber dispersion. We have obtained clear video-rate three-photon images from fluorescent beads, and further optimization toward deep-tissue GFP imaging will be discussed.

 

Without additional optical delay line and complicated dispersion compensation, we investigated the possible fiber-optic nonlinear process to realize simultaneous multicolor two-photon fluorescence imaging. Precise generation of the spectral broadening enabled by self-phase modulation and dispersive wave generation delivers two fully compressible femtosecond pulses, and the relative delay between the pulses can be controlled under different input conditions. Based on a 24MHz Cr:forsterite laser and proper fiber-optic nonlinear conversion, we obtained wide spectral coverages of 900-1200 nm and 700-1200 nm using different fibers. We also demonstrated simultaneous video-rate observation of cyan, green, and red fluorescent beads with controllable sum-frequency excitation.

 

In our system, temporal focusing multiphoton excitation microscopy (TFMPEM), which can excite a large area at once, also can provide high frame rates capability, and is faster than using point-scanning method. But with this characteristic, it cannot excite all area equally distributed, and the scattering problem in bio-tissue is the deeper the worse. So we registered and restored TFMPEM images by using 3D U-Net architecture via the point-scanning images as the ground truth. This study demonstrated 100 frame rates and improved image quality from 8.7 dB to 32.7 dB.

 

Digital image stitching/mosaicking is a basic requirement for a state-of-the-art nonlinear optical mesoscope (NLOM) while imaging a centimeter-scale tissue sample, especially when the NLOM field-of-view (FOV) is limited, owing to a moderate or high numerical aperture (NA) objective lens. For an enhanced effective scanning speed, such stitching operations are expected to be real-time so as to eradicate a need of post-acquisition data processing. We report a post-processing-free sub-minute multicolor gigapixel NLOM imaging technique capable of point-scanning an ultra-large tissue sample at a >30 M/s effective pixel rate while securing a submicron digital resolution.

 

Investigating hematoxylin and eosin (H&E) stained images of frozen-sectioned biopsy tissues is a state-of-the-art clinical diagnosis protocol to assess tumor borders during brain surgery. However, a single assessment usually takes at least 30 minutes and actual margins can be lost owing to the process of frozen sectioning. We report a method to rapidly assess glioma surgery borders, where a whole-mount H&E staining protocol is first applied prior to the slide-free nonlinear mesoscope imaging. The protocol greatly enhances the signal-to-noise ratio and image contrast, and helps maximize the effective scanning rate with much reduced imaging time for centimeter square area sizes.

 

Overcoming photodamage has been challenging in bio-imaging. We demonstrated a power-enhancement model in harmonic generation microscopy by deep learning. With the image acquired at low optical power, the model outputs an image at higher optical power close to the ground-truth image. Consequently, the risk of photo-damage can be reduced.