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 cm 2 area in 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. Plain language summary 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 tum