MUSE: A New, Fast, Simple Microscopy Method for Slide‐Free Histology and Surface Topography

Abstract

IntroductionConventional brightfield or fluorescence microscopy works best with thin, stained specimens mounted on glass slides, but to prepare these requires hours of processing and the help of highly skilled technical personnel. We describe a new, inexpensive form of light microscopy that can generate high‐quality histology and histopathology images directly from cut surfaces of fresh (or fixed) tissue samples of any thickness, with less than 1 minute of preparation.BackgroundWhile alternative microscopy‐scale methods, such as confocal or two‐photon, or optical coherence tomography approaches, can generate reasonable images from thick tissues, the instrumentation is typically complex, expensive and not widely available. We are developing an alternative technique, MUSE (Microscopy with UV Surface Excitation), that relies on simple properties of light, tissue, and fluorescent stains, and can be implemented at relatively low cost. Thick pieces of tissue obtained by biopsy, surgery or necropsy can be imaged directly. The method is non‐destructive, and eliminates requirement for conventional histology processing, formalin fixation, paraffin‐embedding, or thin‐sectioning.MethodUltraviolet light at about 280 nm from an LED arranged to provide oblique illumination, is used to excite just the surface layer of tissue that has been briefly (~10 seconds) stained with common and inexpensive fluorescent dyes. Unlike light of longer wavelength, 280‐nm light only penetrates to a depth of 10 microns or less, and thus excites fluorescent signals, conveniently in the visible range, only from the cut specimen surface. The images, which can be diffraction‐limited, are captured using conventional microscope optics and a standard color cameraResultsMUSE samples from fresh (or fixed) tissues stained with general tissue stains such as rhodamine and Hoechst generate high‐resolution fluorescence images that can be converted in real time from fluorescence to H&E‐like brightfield appearance, for ease of interpretation. However, fluorescence display mode preserves surface shading and depth cues that allow for appreciation of surface profiles important in understanding 3D organization of complex specimens. We have obtained high‐quality MUSE images from normal and diseased tissues from multiple organisms, as well as from plants, and manufactured materials. Moreover, surface profile information provides views beyond what can be seen with in standard thin‐sectioned material, generating results that can resemble those obtainable with scanning electron microscopy (SEM).ConclusionWe have developed a new form of optical microscopy that generates diagnostic‐quality histological images, with enhanced content, from fresh or fixed, but unsectioned tissue, rapidly, with high resolution, simply and inexpensively. We anticipate that there could be widespread adoption in preclinical and clinical research facilities, as well as hospital‐based and stand‐alone medical settings.