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© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement. The ability to histologically assess surgical specimens in real-time is a longstanding 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.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement. The ability to histologically assess surgical specimens in real-time is a longstanding 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.

We present a portable multiphoton system designed for evaluating centimeter-scale surgical margins on surgical breast specimens in a clinical setting. The system is designed to produce large field of view images at a high frame rate, while using GPU processing to render low latency, video-rate virtual H&E images for real-time assessment. The imaging system and virtual H&E rendering algorithm are demonstrated by imaging unfixed human breast tissue in a clinical setting.

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 applicat ions. 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., National Institutes of Health (U.S.) (Grant CA178636-04), National Institutes of Health (U.S.) (Grant CA075289-19), National Institutes of Health (U.S.) (Grant CA183400-02), United States. Air Force Office of Scientific Research (Grant FA9550-12-1-0551), United States. Air Force Office of Scientific Research (Grant FA9550-15-1-0473)

We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.

Rapid histopathological examination of surgical specimen margins using fluorescence microscopy during breast conservation therapy has the potential to reduce the rate of positive margins on postoperative histopathology and the need for repeat surgeries. To assess the suitability of imaging modalities, we perform a direct comparison between confocal fluorescence microscopy and multiphoton microscopy for imaging unfixed tissue and compare to paraffin-embedded histology. An imaging protocol including dual channel detection of two contrast agents to implement virtual hematoxylin and eosin images is introduced that provides high quality imaging under both one and two photon excitation. Corresponding images of unfixed human breast tissue show that both confocal and multiphoton microscopy can reproduce the appearance of conventional histology without the need for physical sectioning. We further compare normal breast tissue and invasive cancer specimens imaged at multiple magnifications, and assess the effects of photobleaching for both modalities using the staining protocol. The results demonstrate that confocal fluorescence microscopy is a promising and cost-effective alternative to multiphoton microscopy for rapid histopathological evaluation of ex vivo breast tissue., National Institutes of Health (U.S.) (Grant R01-CA178636-02), National Institutes of Health (U.S.) (Grant R01-CA075289-18), National Institutes of Health (U.S.) (Grant F32-CA183400-03), United States. Air Force. Office of Scientific Research (Grant FA9550-12-1-0551), United States. Air Force. Office of Scientific Research (Grant FA9550-15-1-0473)