Slide-Free Microscopy via UV Surface Excitation

Abstract

Microscopic evaluation of tissue remains the definitive diagnostic procedure in the evaluation of most solid tumors, as well as of many other disease processes. Conventional 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, based on UV surface excitation (MUSE), that can generate highquality histology and histopathology images directly from cut surfaces of fresh (or fixed) tissue samples of any thickness, with less than 1 minute of preparation. Unlike standard diagnostic processes, it is nondestructive, preserving the specimen for downstream molecular analyses. Preliminary description of this method, which depends on the shallow tissue penetration of sub-300-nm light, can be found in [1].While alternative microscopy-scale methods, such as confocal, two-photon, or optical coherence tomography approaches [2-6], can generate reasonable images from thick tissues, such instrumentation is typically complex, expensive, and not widely available. With MUSE, thick pieces of tissue obtained by biopsy, surgery or necropsy can be imaged directly, eliminating any requirement for conventional histology processing, formalin-fixation, paraffin-embedding, or thin-sectioning. The method is rapid, and by providing diagnostic-level information in minutes, can potentially change the velocity of clinical encounters; Moreover, MUSE can be more informative than conventional histology, because it provides information on surface topography, typically effaced by thin-sectioning procedures. Figure 1 illustrates the basic MUSE configuration, which is based on an inverted microscope design, largely because it is the easiest way to position large, variably shaped tissue specimens. The sample rests on (or is lightly compressed against) a UV-transmitting flat optical surface, typically quartz or sapphire (replacing the coverslip in conventional microscopy). The excitation light is provided by one or more UV-emitting LEDs with maximum emission wavelength centered around 275-285 nm, arranged to illuminate the sample in oblique configuration. We typically use a 10X lens and a scientific-grade 9-Mpixel color (RGB) CCD camera.Samples need only be cut with a sharp knife or scalpel to provide a flat imaging surface, and then briefly stained. A convenient dye combination includes rhodamine and Hoechst in phosphate-buffered saline. The specimen is dipped into this mixture for 10 s and then washed gently in more PBS, and then can be immediately positioned on the UV-transparent stage for imaging. Depending on the specimen and the brightness of the LED(s) used, images can be acquired in as little as 100 ms per field. Multiple fields can be taken by translating the stage, and then montaged into a single, large field-of-view final image file.MUSE images are acquired in fluorescence and take advantage of the fact that many fluorescent dyes can all be ...

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“…In addition to confocal [3,5,6] and LSFM mentioned above, multiple other imaging methods have also been utilized for non-destructive imaging of tissues, including optical coherence tomography (OCT) [7], microscopy with UV surface excitation (MUSE) [8,9], structured illumination microscopy (SIM) [10], and nonlinear microscopy [11,12]. While these methods may have significant cost, complexity, and speed advantages for 2D imaging compared to LSFM, for 3D tissue imaging they are limited by varying combinations of low resolution, low penetration depth, or a relatively slow imaging speed.…”