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Tissue biopsy and histological evaluation is the gold standard for disease diagnosis including cancer. For example, a punch several millimeters in diameter is often used to biopsy suspicious skin sites. The biopsy is then formalin fixed, paraffin embedded, sectioned, stained with hematoxylin and eosin (H&E), and examined by a pathologist. While this process has been the gold standard for decades, two limitations are recognized. First, the biopsy is invasive with a limited number that can be reasonably tolerated by the patient. Second, the tissue processing steps are slow. We report an alternative approach consisting of a laser microbiopsy for harvest of sub-microliter (<1 mm3) tissue sections combined with rapid virtual H&E staining methods. A Ho:YAG laser (Lumenis P120) was shaped into an annular beam and focused onto ex vivo porcine skin. The epidermis and dermis were laser cut and the tissue section in the center of the annulus was ejected and collected by an overlying glass coverslip. Tetrafluoroethane (R134A) was sprayed at the ablation site prior to ablation and at the collected tissue section post ablation to limit thermal damage and preserve histological features. Two virtual H&E imaging methods were tested with confocal microscopy. The first combined acridine orange fluoresce with reflectance. The second combined acridine orange and sulforhodamine 101 fluorescence. For each method, the two channels were false colored and combined to create virtual H&E images. Virtual H&E images show histological features, including cell nuclei. Laser microbiopsy is minimally invasive, harvesting tissue sections on the order of 0.01 to 0.1 mm3, and tissue processing is rapid requiring 2 min or less for staining. Laser microbiopsy is a promising candidate technique for rapid minimally invasive diagnosis.
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