Label-free multi-photon imaging has been a powerful tool for studying tissue microstructures and biochemical distributions, particularly for investigating tumors and their microenvironments. However, it remains challenging for traditional bench-top multi-photon microscope systems to conduct ex vivo tumor tissue imaging in the operating room due to their bulky setups and laser sources. In this study, we designed, built, and clinically demonstrated a portable multi-modal nonlinear label-free microscope system that combined four modalities, including two- and three- photon fluorescence for studying the distributions of FAD and NADH, and second and third harmonic generation, respectively, for collagen fiber structures and the distribution of micro-vesicles found in tumors and the microenvironment. Optical realignments and switching between modalities were motorized for more rapid and efficient imaging and for a light-tight enclosure, reducing ambient light noise to only 5% within the brightly lit operating room. Using up to 20 mW of laser power after a 20x objective, this system can acquire multi-modal sets of images over 600 μm × 600 μm at an acquisition rate of 60 seconds using galvo-mirror scanning. This portable microscope system was demonstrated in the operating room for imaging fresh, resected, unstained breast tissue specimens, and for assessing tumor margins and the tumor microenvironment. This real-time label-free nonlinear imaging system has the potential to uniquely characterize breast cancer margins and the microenvironment of tumors to intraoperatively identify structural, functional, and molecular changes that could indicate the aggressiveness of the tumor.
Yi Sun, Sixian You, Haohua Tu, Darold R. Spillman, Marina Marjanovic, Eric J. Chaney, George Z. Liu, Partha S. Ray, Anna Higham, and Stephen A. Boppart, "Intra-operative label-free multimodal multiphoton imaging of breast cancer margins and microenvironment (Conference Presentation)," Proc. SPIE 10043, Diagnosis and Treatment of Diseases in the Breast and Reproductive System, 1004307 (Presented at SPIE BiOS: January 28, 2017; Published: 19 April 2017); https://doi.org/10.1117/12.2251057.5370007617001.
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