Abstract
Microscopy with ultraviolet surface excitation (MUSE) typically has an optical sectioning thickness significantly larger than standard physical sectioning thickness, resulting in increased background fluorescence and higher feature density compared to formalin-fixed, paraffin-embedded physical sections. We demonstrate that high-index immersion with angled illumination significantly reduces optical sectioning thickness through increased angle of refraction of excitation light at the tissue interface. We present a novel objective dipping cap and waveguide-based MUSE illuminator design with high-index immersion and quantify the improvement in optical sectioning thickness, demonstrating an e-1 section thickness reduction to 6.67 µm in tissue. Simultaneously, the waveguide illuminator can be combined with high or low magnification objectives, and we demonstrate a 6 mm2 field of view, wider than a conventional 10x pathology objective. Finally, we show that resolution and contrast can be further improved using deconvolution and focal stacking, enabling imaging that is robust to irregular surface profiles on surgical specimens.
Highlights
Conventional formalin-fixed, paraffin-embedded (FFPE) histology, based on physical sectioning of tissue specimens into optically thin slices, remains the current standard of care for assessment of surgical specimens, biopsies, and most excised tissue specimens
We demonstrate the use of a high-index immersion objective dipping cap and waveguide-based illuminator to decrease the optical sectioning thickness and characterize the improvement in resolution
With angled illumination (Fig. 2(b)), the incident deep ultraviolet (DUV) photons still travel, on average, one mean free absorption path before absorption but since the path is angled relative to the tissue surface, the optical sectioning thickness becomes the tangential component of the mean free absorption path
Summary
Conventional formalin-fixed, paraffin-embedded (FFPE) histology, based on physical sectioning of tissue specimens into optically thin slices, remains the current standard of care for assessment of surgical specimens, biopsies, and most excised tissue specimens. FS processing takes a shorter time (20-30 minutes) by replacing paraffin embedding with rapid freezing, but is prone to freezing artifacts and difficult to perform on many tissue types [1,2], while requiring expensive equipment with highly skilled technicians to perform delicate sectioning. Recent developments in microscopy have given rise to techniques that replace physical sectioning with optical sectioning, extracting virtual histology sections from a larger 3D tissue specimen with little to no processing. These new techniques dramatically reduce time and skill requirements, and are attractive alternatives to FS
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