Intelligent Microtome Instrumentation

描述（由申请人提供）：在整个生物体中定位荧光标记的细胞对于基因治疗、干细胞治疗递送方法的开发和药物研究是重要的。该项目将开发一种智能切片机，以识别整个小鼠中的荧光标记细胞，并将这些细胞收集在薄切片中。在第一阶段，一个小哺乳动物冷冻切片机与基于LED的组织照明荧光成像将被构建。几个CCD相机将进行评估，以建立空间分辨率和灰度分辨率需要提供必要的荧光成像能力的仪器。该仪器将在分离的肌肉、含有绿色荧光蛋白（GFP）标记细胞的组织匀浆和整只小鼠上进行测试。这些样本中表达GFP的细胞将为II期下一代仪器的开发提供数据。 第二阶段将增加使用荧光图像数据的功能，以便仅在检测到荧光细胞时智能地获取薄切片。在切片过程中获得的荧光图像将提供形态学指导，以通过激光捕获显微切割来识别和分离固定切片中的相同细胞。自动化薄组织采集将是一个额外的能力，并将与智能荧光分析集成，以产生一个完全自动化的仪器。该仪器将允许对整个动物进行处理，以获得用于激光解剖的薄切片，并用于选定组织区域的生化和/或PCR后处理。

DESCRIPTION (provided by applicant): Locating fluorescently-tagged cells in whole organisms is important to the development of gene therapy, stem cell therapy delivery methods, and for drug research. This project will develop an intelligent microtome to identify fluorescently labeled cells in whole mice and to collect those cells in thin sections. During Phase I, a small-mammal cryomicrotome with LED-based tissue illumination for fluorescence imaging will be constructed. Several CCD cameras will be evaluated to establish the spatial resolution and gray-scale resolution needed to provide necessary fluorescence-imaging capabilities for the instrument. The instrument will be tested on isolated muscle, tissue homogenates containing green fluorescent protein (GFP) labeled cells, and on whole mice. The cells expressing GFP in these samples will provide data for the development of next-generation instrumentation during Phase II. Phase II will add capabilities to use the fluorescence image data to intelligently acquire thin sections only when fluorescent cells are detected. Fluorescence images acquired during sectioning will provide morphological guides to identify and isolate the same cells in mounted sections by laser-capture microdissection. Automated thin-tissue acquisition will be an added capability and will be integrated with intelligent fluorescence analysis to produce a completely automated instrument. The instrument will allow processing of whole animals to obtain thin sections for laser dissection and for biochemical and/or PCR post processing of select tissue regions.