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

Abstract

The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.

Evaluation of silicon photomultipliers for multiphoton and laser scanning microscopy

“…Video-rate frames were recorded while the specimen was examined in real time, simulating a workflow where a surgical margin was evaluated for the presence of cancer. Frames were then stitched into a mosaic using a method described previously 8 and rendered as virtual hematoxylin and eosin slides. 15 Imaging a localized bright region with infiltrative basal cell carcinoma resulted in saturation of both detectors (Fig.…”

Polarization‐dependent second‐harmonic generation for collagen‐based differentiation of breast cancer samples

“…Several reports have provided important information for both cells and tissues during inflammation and cancer upon application of these nondestructive techniques. [1][2][3][4] A previous study from our group demonstrated that THG could evaluate the density and size of tumor cells within malignant tissues and suggested the use of such technology in cancer prognosis. 5 Recently, this technology has been applied on live tissue with very promising results and has therefore been proposed for clinical studies.…”

Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins

“…Optical-sectioning microscopy techniques can potentially enable rapid nondestructive imaging of surgical margin surfaces to guide tumor-resection procedures. For instance, confocal microscopy, 13 – 15 nonlinear microscopy, 16 – 20 open-top light-sheet (OTLS) microscopy, 21 structured-illumination microscopy (SIM), 22 – 24 photoacoustic microscopy (PAM), 25 and optical coherence tomography (OCT), 26 , 27 among others, have been shown to enable high-resolution imaging of freshly excised surgical specimens that can approach the quality of gold-standard histology. All of these techniques involve trade-offs between speed, simplicity, cost, resolution, and image contrast/depth.…”