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. METHODS: We develop a training-free true-H&E Rapid Fresh digital-Pathology (the-RFP) technique which is 4 times faster than the conventional preparation of frozen sections. The-RFP is assisted by a mesoscale Nonlinear Optical Gigascope (mNLOG) platform with a streamlined rapid artifact-compensated 2D large-field mosaic-stitching (rac2D-LMS) approach. A sub-6-minute True-H&E Rapid whole-mount-Soft-Tissue Staining (the-RSTS) protocol is introduced for soft/frangible fresh brain specimens. The mNLOG platform utilizes third harmonic generation (THG) and two-photon excitation fluorescence (TPEF) signals from H and E dyes, respectively, to yield the-RFP images. RESULTS: We demonstrate the-RFP technique on fresh excised human brain specimens. The-RFP enables optically-sectioned high-resolution 2D scanning and digital display of a 1 cm2 area in <120 seconds with 3.6 Gigapixels at a sustained effective throughput of >700 M bits/sec, with zero post-acquisition data/image processing. Training-free blind tests considering 50 normal and tumor-specific brain specimens obtained from 8 participants reveal 100% match to the respective formalin-fixed paraffin-embedded (FFPE)-biopsy outcomes. CONCLUSIONS: We provide a digital ITA solution: the-RFP, which is potentially a fast and reliable alternative to FS-pathology. With H&E-compatibility, the-RFP eliminates color- and morphology-specific additional interpretation training for a pathologist, and the-RFP-assessed specimen can reliably undergo FFPE-biopsy confirmation.

Brain tumors can be fatal and surgery is often required to remove them. During surgery, clinicians need to look for any leftover tumor tissue so that recurrence of the disease can be avoided. This requires sectioning of frozen tissue samples, staining them, and visualizing structural details under a microscope in the lab. This process should be fast to make the operation shorter and safer for the patient. Here, we provide an alternative approach to staining and imaging tumor samples, which is much faster than the current process. We show that our approach works with fresh tumor samples, avoiding the need to freeze and physically section them. We can distinguish normal versus tumor tissues, and pathologists do not require special training to use our approach. Our approach might ultimately help to improve the speed, safety, and outcomes of brain tumor surgery.

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. For complete details on the use and execution of this protocol, please refer to Borah et al. (2021).

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. We report a digital approach called rapid denoised contrast enhancement (DCE), which digitally mimics a hardware-based adaptive/controlled illumination technique by means of digitally optimizing the signal strengths and hence the visibility of such weak-intensity structures while mostly preventing the saturation of the brightest ones. With large field-of-view (FOV) two-photon excitation fluorescence (TPEF) neuronal imaging, we validate the effectiveness of DCE over state-of-the-art digital image processing algorithms. With compute-unified-device-architecture (CUDA)-acceleration, a real-time DCE is further enabled with a reduced time complexity.

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. Here, we propose and demonstrate a single-laser-based four-wavelength excitation source for two-photon fluorescence microscopy. Using a sub-100 fs 1,070-nm Yb:fiber laser to pump an ultrashort nonlinear photonic crystal fiber in the low negative dispersion region, we introduced efficient self-phase modulation and acquired a blue-shifted spectrum dual-peaked at 812 and 960 nm with 28.5% wavelength conversion efficiency. By compressing the blue-shift near-IR spectrum to 33 fs to ensure the temporal overlap of the 812 and 960 nm peaks, the so-called sum frequency effect created the third virtual excitation wavelength effectively at 886 nm. Combined with the 1,070 nm laser source as the fourth excitation wavelength, the all-fiber-format four-wavelength excitation source enabled simultaneous four-color two-photon imaging in Brainbow AAV-labeled (TagBFP, mTFP, EYFP, and mCherry) brain samples. With an increased number of excitation wavelengths and improved excitation efficiency than typical commercial femtosecond lasers, our compact four-wavelength excitation approach can provide a versatile, efficient, and easily accessible solution for multiple-color two-photon fluorescence imaging in the field of neuroscience, biomolecular probing, and clinical applications with at least four spectrally-distinct fluorophores.

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 aliasing. We introduce a Nyquist figure-of-merit (NFOM) parameter to characterize a laser-scanning MPM in terms of its optical-resolution retrieving ability. Based on NFOM, we define the maximum aliasing-free FOV, and subsequently, a cross-over excitation wavelength, below which the FOV becomes NFOM-constrained irrespective of an optimized optical design. We validate our idea in a custom-built mesoscopic-MPM with millimeter-scale FOV yielding an ultra-high FOV-resolution ratio of >3,000, while securing up-to a 1.6 mm Nyquist-satisfied aliasing-free FOV, a ∼400 nm lateral resolution, and a 70 M/s effective voxel-sampling rate, all at the same time.