RESUMEN

Advanced fluorescence imaging modalities such as confocal microscopy and two photon fluorescence microscopy can provide rapid, real-time histology images, but the mounting of fresh tissue specimens in standard orientations required for diagnosis without embedding and sectioning remains an unsolved problem. Here, we introduce a piston-based specimen holder designed for consistent, even pressure distribution. We improve upon previous designs by incorporating an air piston system with a flexible membrane and wick that extracts fluid during compression. We combine this with support fixtures to aid in the distribution of pressure, enabling imaging of specimens with small surface areas relative to their thickness, such as bisected shave skin biopsies in standard orientation without embedding or sectioning. We image both fresh biopsy specimens and diagnostic Mohs first stage specimens during clinical procedures, demonstrating improved visualization of the tissue surface in real time. Finally, we show that conventional cryosectioning can exaggerate the extent of margin positivity, which can be avoided using the piston-based holder.