Rapid digital pathology of H&E-stained fresh human brain specimens as an alternative to frozen biopsy.

Background: Hematoxylin and Eosin (H&E)-based frozen section (FS) pathology is presently the global standard for intraoperative tumor assessment (ITA). Preparation of frozen section is labor intensive, which might consume up-to 30 minutes, and is susceptible to freezing artifacts. An FS-alternative technique is thus necessary, which is sectioning-free, artifact-free, fast, accurate, and reliably deployable without machine learning and/or additional interpretation training.

Construction of a high-NFOM multiphoton microscope with large-angle resonant raster scanning.

A resonant-scanning multiphoton optical microscope (MPM) with a millimeter-scale field-of-view (FOV) often encounters a poor Nyquist figure-of-merit (NFOM), leading to an aliasing effect owing to limited effective voxel-sampling rate. In this protocol, we provide a design guideline to enable high-NFOM MPM imaging while simultaneously securing a large FOV/digital-resolution ratio and a fast resonant raster-scanning speed. We further provide a free version of our custom acquisition software to assist with a smooth and easy construction process.

A rapid denoised contrast enhancement method digitally mimicking an adaptive illumination in submicron-resolution neuronal imaging.

Optical neuronal imaging often shows ultrafine structures, such as a nerve fiber, coexisting with ultrabright structures, such as a soma with a substantially higher fluorescence-protein concentration. Owing to experimental and environmental factors, a laser-scanning multiphoton optical microscope (MPM) often encounters a high-frequency background noise that might contaminate such weak-intensity ultrafine neuronal structures. A straightforward contrast enhancement often leads to the saturation of the brighter ones, and might further amplify the high-frequency background noise.

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability.

The Nyquist-Shannon criterion has never been realized in a laser-scanning mesoscopic multiphoton microscope (MPM) with a large field-of-view (FOV)-resolution ratio, especially when employing a high-frequency resonant-raster-scanning. With a high optical resolution nature, a current mesoscopic-MPM either neglects the criterion and degrades the digital resolution to twice the pixel size, or reduces the FOV and/or the raster-scanning speed to avoid . We introduce a Nyquist figure-of-merit (NFOM) parameter to characterize a laser-scanning MPM in terms of its optical-resolution retrieving ability.

Single-laser-based simultaneous four-wavelength excitation source for femtosecond two-photon fluorescence microscopy.

Multicolor labeling of biological samples with large volume is required for omic-level of study such as the construction of nervous system connectome. Among the various imaging method, two photon microscope has multiple advantages over traditional single photon microscope for higher resolution and could image large 3D volumes of tissue samples with superior imaging depth. However, the growing number of fluorophores for labeling underlines the urgent need for an ultrafast laser source with the capability of providing simultaneous plural excitation wavelengths for multiple fluorophores.