3D Microscopy with Ultraviolet Surface Excitation (3D-MUSE) Summary. We will create powerful techniques for performing 3D histology-quality imaging of small to large volume specimens (e.g., entire mouse or human organ) with extraordinary contrasts to address a very large number of preclinical, biotechnology, and clinical applications. We will combine Microscopy with Ultraviolet Sur- face Excitation (MUSE) (invented at Lawrence Livermore National Laboratory and extended at University of California at Davis), with 3D serial sectioning and block face imaging technologies (developed at Case West- ern Reserve University), to implement 3D-MUSE. Briefly, MUSE fluorescence imaging uses excitation at wave- lengths shorter than 300 nm, a range at which tissue penetration is limited to a few microns deep. This allows one to image?at sub-nuclear resolution?only the very surface of a piece of tissue, making it ideal for serial block-face imaging. Many common histological stains, autofluorescence, and fluorescent proteins excite at these sub-300-nm wavelengths and emit at their familiar wavelengths in the visible range. Because the excita- tion range is distant from emitted signals, images can be captured using a color camera, without additional fil- ters, providing a broad and informative color palette, beyond what is obtained using narrow-band-filter sys- tems. With color-sensitive volume rendering, and combined color and morphology-based segmentation, 3D microanatomy and pathology will be visualized and quantified. Image-guided histology will allow 2D/3D live- time monitoring to guide optional section acquisition for molecular studies using immunohistochemistry and manual or laser-capture micro-dissection. When fully realized, potential applications, out of many, include 3D microanatomy, mouse phenotyping, embryo cell lineage tracking, monitoring therapeutic nanoparticle delivery, as well as studies of cancer physiology, cancer immunotherapy, stem cells, toxicology, and biology. In all of these cases, we anticipate that there will be great value added by the third dimension. We will build on exper- tise in MUSE and block-face imaging systems to create test beds for evaluating 3D-MUSE. We will optimize tissue immobilization, staining, and imaging, and advance MUSE-specific software for 3D segmentation, visu- alization, and analysis. If our research is successful, we will transfer 3D-MUSE technology to commercial enti- ties that can create products for researchers across the world. It will likely be possible to create inexpensive, highly informative 3D-MUSE imaging systems that will empower many applications. Ultimately, the number of researchers using 3D-MUSE around the world will define project success.
We will create simple, accessible and powerful techniques for performing 3D histology-quality imaging of small to large volume specimens to serve a large number of preclinical, biotechnology, and clinical applications.
