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

Tadayuki Yoshitake,Beverly E Faulkner-Jones,James G Fujimoto,Liza M Quintana,Michael G Giacomelli,Hilde Vardeh,James L Connolly,Daihung Do,Lucas C Cahill

doi:10.1038/s41598-018-22264-2

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.

Highlights

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment
We further demonstrate that the transverse image resolution of Microscopy with ultraviolet surface excitation (MUSE) is limited by the optical sectioning thickness and use co-registered nonlinear microscopy (NLM) to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination
We designed a dual camera fluorescence imaging module which attached to the microscope eyepiece port of a commercial NLM system (Fig. 1b) and mounted 3D printed deep ultraviolet (DUV) illuminators on the objective (Fig. 1c,d), enabling water immersion MUSE imaging co-registered to NLM

Summary

Introduction

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Various methods of histological imaging using optical sectioning microscopy have been proposed, including confocal fluorescence microscopy (CFM)[2,3,4], two-photon excitation nonlinear microscopy (NLM)[5,6,7], structured illumination microscopy (SIM)[8,9], optical coherence tomography (OCT)[10,11,12], and light sheet microscopy[13]. These techniques enable rapid evaluation of pathology with high correspondence to standard histological techniques[2,5]. OCT provides high imaging rates and deep penetration into tissue, but is incompatible with fluorescent stains and has limited ability to visualize cell nuclei which are important diagnostic features

Methods

Results

Conclusion