Current microscopy-based tissue diagnostics, particularly hematoxylin and eosin (H&E) histology, requires multiple complex tissue processing steps: fixation, paraffin embedding, microtome sectioning, dying the tissue, and imaging individual slides through a bright field microscope. The time and labor-intensive result of this process makes it unsuitable for patient point-of-care evaluation. Therefore, many bedside procedures are completed without efficient real-time analysis of tissue adequacy and diagnostic results are unnecessarily delayed. Additionally, research experiments that require information regarding changes to tissue morphology or function before proceeding to the next experimental phase are severely interrupted by histology processing in their workflow. Fluorescence histology, which relies on rapid fluorescent staining of tissue, optical sectioning microscopy, and image processing for digital viewing, can provide an inexpensive, non-destructive, 3-dimensional, and fast alternative to traditional histology and point-of-care screening protocols. The objective of this work is to further advance the concept of “fluorescence histology,” in which traditional histopathology preparation methods are replaced by optical-sectioning (in lieu of physical sectioning), sensitive and flexible fluorescence-based contrast (in lieu of chromophore-based contrast), and computational strategies to replicate traditional color-schemes. In this work, we demonstrate the development and use of a fluorescent analogue to H&E on fixed and frozen tissue sections and fresh human biopsies. This fluorescent analogue, DRAQ5 & eosin, is compared against the current single-agent, monochrome fluorescence histology system, and their effects on diagnostic downstream molecular analyses, including quantitative-PCR and immunohistochemistry, is evaluated. We create a methodology to develop and characterize fluorescent analogues for any histological stain, with demonstration using Masson’s Trichrome and Periodic Acid-Schiff, enabling the expansion of fluorescence histology for multiple applications. This work demonstrates the ability to improve point-of-care pathology and research by replacing destructive, incomplete, and time-consuming histology with fluorescence histology, which preserves the tissue for later analysis or experiments while providing accurate and rapid histology assessment.
