3D Microscopy with Ultraviolet Surface Excitation (3D-MUSE)

紫外光表面激发三维显微技术(3D-MUSE) 总结。我们将创造强大的技术来执行3D组织学质量的从小到大的成像 体积标本(例如，整个小鼠或人体器官)，具有非凡的对比度，以处理非常大的 临床前、生物技术和临床应用的数量。我们将把显微技术和紫外光技术结合起来 面孔激发(MUSE)(由劳伦斯·利弗莫尔国家实验室发明，在华盛顿大学推广 加州戴维斯分校)，采用3D连续切片和块面成像技术(在凯斯韦斯特开发- Ern Reserve University)，以实现3D-MUSE。简而言之，缪斯荧光成像使用的是波激发- 长度小于300纳米，在这个范围内，组织渗透被限制在几微米深。这使得 一种是以亚核分辨率成像，只成像一块组织的最表面，这使其成为连续成像的理想选择 块面成像。许多常见的组织学染色、自发荧光和荧光蛋白在 这些波长低于300纳米，并在可见光范围内以它们熟悉的波长发射。因为兴奋的是- 距离发射信号很远，可以使用彩色摄像机拍摄图像，而不需要额外的文件- TERS提供了一个宽广且信息丰富的调色板，超越了使用窄带滤镜系统所能获得的效果。 Temes.通过颜色敏感体绘制，并结合基于颜色和形态的分割，3D 显微解剖和病理学将被可视化和量化。图像引导的组织学将允许2D/3D实时- 使用免疫组织化学和免疫组织化学技术指导分子研究的可选切片获取的时间监控 手动或激光捕获显微解剖。当完全实现时，潜在的应用程序包括3D 显微解剖学，小鼠表型，胚胎细胞谱系追踪，监测治疗性纳米颗粒输送， 以及癌症生理学、癌症免疫疗法、干细胞、毒理学和生物学的研究。在所有的 在这些情况下，我们预计第三维度将会有很大的附加值。我们将在Exper的基础上- 在缪斯和块面成像系统中安装，以创建用于评估3D-MUSE的试验台。我们将优化 组织固定、染色和成像，以及先进的缪斯专用软件，用于3D分割、可视化和 具体化和分析性。如果我们的研究成功，我们将把3D-MUSE技术转移到商业实体-- 可以为世界各地的研究人员创造产品的纽带。很有可能创造出价格低廉、 信息量大的3D-MUSE成像系统将支持许多应用程序。归根结底， 世界各地使用3D-MUSE的研究人员将定义项目的成功。

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.