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Reflection-mode virtual histology using photoacoustic remote sensing microscopy

Histological visualizations are critical to clinical disease management and are fundamental to biological understanding. However, current approaches that rely on bright-field microscopy require extensive tissue preparation prior to imaging. These processes are labor intensive and contribute to delays in clinical feedback that can extend to two to three weeks for standard paraffin-embedded tissue preparation and interpretation. Here, we present a label-free reflection-mode imaging modality that reveals cellular-scale morphology by detecting intrinsic endogenous contrast. We accomplish this with the novel photoacoustic remote sensing (PARS) detection system that permits non-contact optical absorption contrast to be extracted from thick and opaque biological targets with optical resolution. PARS was examined both as a rapid assessment tool that is capable of managing large samples (>1 cm2) in under 10 minutes, and as a high contrast imaging modality capable of extracting specific biological contrast to simulate conventional histological stains such as hematoxylin and eosin (H&E). The capabilities of the proposed method are demonstrated in a variety of human tissue preparations including formalin-fixed paraffin-embedded tissue blocks and unstained slides sectioned from these blocks, including normal and neoplastic human brain, and breast epithelium involved with breast cancer. Similarly, PARS images of human skin prepared by frozen section clearly demonstrated basal cell carcinoma and normal human skin tissue. Finally, we imaged unprocessed murine kidney and achieved histologically relevant subcellular morphology in fresh tissue. This represents a vital step towards an effective real-time clinical microscope that overcomes the limitations of standard histopathologic tissue preparations and enables real-time pathology assessment.