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This invention is an imaging system that enables real-time histological analysis of large surgically removed tissues without the need for physical sectioning. The apparatus includes a primary imaging system, auxiliary imaging system, specimen holder, user input device, processing unit, and a user display device. The primary imaging system is an inverted microscope capable of performing optical depth sectioning and configured to acquire a sequence of images. The auxiliary imaging system functions as a camera to acquire auxiliary images covering an area larger than the area of each image acquired by the primary imaging system. The specimen holder positions the tissue of interest on a transparent window with position sensors which measure detect the tissue’s position and translate spatial information to a focal plane in the auxiliary imaging system. The user input device is configured to accept user input which is translated to the specimen holder. The processing unit utilizes the sequences of images acquired by the imaging systems with the spatial information from the specimen position holder to generate a composite representation of the tissue specimen at the cell nuclei resolution. Finally, the display device displays the composite representation of the tissue specimen from the processing unit in real-time.

Collectively, this technology achieves fast histological analysis of large tissue specimens and identifies margins of pathological tissue within the specimen. The system can implement various optical techniques, including multiphoton microscopy and confocal fluorescence microscopy, or techniques based on structured illumination, to detect fluorescent or scattered light at a magnification comparable to a conventional histology microscope, but at an imaging rate suitable for real-time user evaluation.