Rapid histopathological imaging of skin and breast cancer surgical specimens using immersion microscopy with ultraviolet surface excitation

Abstract

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.

Open-Top Light-Sheet Microscopy Image Atlas of Prostate Core Needle Biopsies

“…21 In contrast, microscopy with ultraviolet surface excitation is inexpensive with remarkably quick imaging speed, making it an attractive technique for rapid fresh tissue imaging, but cannot image below a tissue surface because of the use of ultraviolet wavelengths for illumination. 25 Selection of the optimal technique depends on both the clinical application and the needs/resources of the pathology laboratory. Open-top light-sheet microscopy strikes a balance among resolution, speed of imaging, depth, and cost, making the technology well suited for 3D microscopy of biopsy specimens.…”

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins

“…Because physical sectioning accounts for the vast majority of processing delay for both FFPE and FSA histology, optical sectioning can dramatically reduce processing times and enable intraoperative histological examination in scenarios where FFPE and FSA histology are too time consuming. Various methods have been proposed for imaging breast, prostate and other surgical margins without physical sectioning, including optical coherence tomography (OCT) [18][19][20][21][22], reflectance confocal microscopy (RCM) [23,24], confocal fluorescence microscopy (CFM) [25][26][27][28][29], structured illumination microscopy (SIM) [30], light sheet microscopy [31], microscopy with ultraviolet surface excitation (MUSE) [32,33], and nonlinear microscopy (NLM) [34][35][36][37][38]. Stimulated Raman scattering (SRS) has also been demonstrated for surgical histology [39], but typically has appreciably lower imaging speed or signal to noise ratio when performed without physical sectioning in reflectance mode.…”

FalseColor-Python: a rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology

“…In order to improve throughput, non-destructiveness, sampling extent, and in some cases, to provide 3D information, several slide-free microscopy techniques have recently been explored for use in clinical settings. For example, techniques such as confocal microscopy [1,2], multiphoton microscopy [3][4][5][6], microscopy with UV surface excitation (MUSE) [7][8][9], and structured illumination microscopy (SIM) [10,11] have been explored as slide-free alternatives to frozen-section histology for rapid interoperative guidance.…”