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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 postoperativehistopathology 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 unfixedtissue 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-effectivealternative 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

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue