SUMMARY A common practice in medicine for handling freshly biopsied human tissue is to rapidly put it into a formalin solution. This “fixation” preserves the tissue from decay, allowing transportation to a (remote) pathology laboratory for standard histopathology or other biochemical/genetical analyses. However, formalin instantly “kills” the tissue, so that the biochemical and metabolic functions associated with its vital state are permanently lost. These functions likely contain diagnostic information not available from standard histopathology, e.g. the pristine picture of an active tumor microenvironment before the structural and chemical perturbations known in routine histological sample treatments. Thus, it will be highly beneficial to image “tissue vitality” noninvasively and rapidly (within minutes) before the fixation, by a novel optical imaging technology. Although numerous technologies have been developed for this purpose, their diagnostic capabilities independent from standard histopathology have not been unambiguously demonstrated, due to the lack of a correlative technology to co- register/co-localize the virtual optical sections of vital tissue (optical imaging) and the actual formalin-fixed paraffin-embedded sections of treated/nonvital tissue (standard histopathology). As a result, it is often difficult to justify the additional cost of novel optical imaging technologies in clinical standard-of-care processes. In this project, we aim to develop correlative microscopy between standard histopathology (operated in a linear/single-photon optical regime) and a novel optical imaging technology based on multiphoton processes, i.e. multiphoton pre-histopathology. The latter has been demonstrated in our prior work to visualize multiple unlabeled endogenous biomolecules and segment a rich set of cellular and extracellular components. The proposed correlative microscopy will retrace optical sections of fresh ex vivo tissue that were imaged using the label-free multiphoton pre-histopathology to the corresponding formalin-fixed paraffin-embedded sections with diverse histological stains. For proof-of-concept demonstration (Phase I), we will use discarded fresh rodent tissue specimens to mimic freshly biopsied human tissue specimens, and co-register a wide area (1x1 mm2) of image in the multiphoton pre-histopathology with its counterpart in standard hematoxylin and eosin (H&E) histology. In a future stage (Phase II), we will expand the co-registration area from 1x1 mm2 to 10x10 mm2 to accommodate larger sample sizes, extend the stains from H&E to diverse immunohistological stains, and switch the samples from the meat products to fresh human tissue biopsies in a clinical setting. The successful outcome of this project will equip pathologists with a real-time, label-free, slide-free, digital, and point-of-care or point-of-procedure tool to image “tissue vitality”, without affecting the existing histology workflow but with newly added critical inf...