Confocal enhanced episcopic fluorescent image capture (EFIC)

描述（由申请人提供）：本申请寻求获得共焦增强型反射荧光图像捕获（EFIC）系统。EFIC成像需要在石蜡包埋标本的切割表面捕获组织荧光。当使用具有固定照片位置的切片机切割每个切片时，捕获暴露在块面处的样本的落射荧光图像。鉴于照片位置是不变的，所收集的2D图像堆栈是完美配准的，因此可以在任何成像平面中进行数字切除。它也可以快速重建，以生成高分辨率的3D渲染的标本。在第一代EFIC成像系统中，使用标准汞照明通过落射荧光成像捕获表面荧光。为了防止荧光从块表面下渗出，苯胺，一种光阻挡染料通过费力且困难的高温包埋过程被掺入石蜡中。有了这个建议，我们将组装第二代EFIC成像系统，将免除高温石蜡包埋苯胺的需要。成像将使用Leica宽视场超级变焦LSI共焦显微镜进行。由于共聚焦成像将允许仅捕获表面荧光，因此它将消除在石蜡中掺入苯胺染料的需要。这使得任何石蜡包埋的标本都可以进行EFIC成像处理。此外，使用适当的激光器和激发/发射滤光片，EFIC成像可以与使用荧光蛋白标记的表达构建体跟踪特定细胞谱系相结合。总的来说，EFIC成像比MRI获得的图像具有更好的分辨率，并且具有大多数研究人员可以轻松学习的优势，因为它本质上是一种与共聚焦成像相结合的组织学技术。在任何成像平面中数字切除2D图像堆栈的能力以及快速3D重建的简易性对于评估和诊断解剖畸形（例如与先天性心脏病相关的解剖畸形）或血管图案或分支形态发生中的缺陷是非常宝贵的。此外，可以使用完美配准的2D图像堆栈或3D重建来执行解剖结构的定量评估，例如血管/小管尺寸或其分支复杂性，或者腔室管腔尺寸的大小或组织厚度。该共聚焦增强型EFIC系统将使用现成的设备Leica SM 2500切片机和Leica LSI共聚焦显微镜进行组装。自定义所需的贴附和对齐共聚焦显微镜的照片位置的切片机已经开发了慷慨的种子资金从医学院和借用LSI共聚焦显微镜从发育生物学系。因此，本提案中要求的资金将允许立即安装专用的共焦EFIC成像系统。这将极大地增强六名资金充足的NIH研究人员的研究计划，填补了高分辨率组织结构分析的2D/3D成像技术的空白。 公共卫生相关性：本申请寻求获取将提供三维解剖结构的详细可视化的整个组织成像系统。这将有助于检测与出生缺陷和其他获得性疾病相关的组织结构变化，并允许对解剖和组织缺陷进行定量分析，以产生对疾病机制的新见解。

DESCRIPTION (provided by applicant): This application seeks to acquire a confocal enhanced episcopic fluorescence image capture (EFIC) system. EFIC imaging entails the capture of tissue fluorescence at the cut surface of paraffin embedded specimen. As each section is cut using a microtome with a fixed photo position, an epifluorescence image of the specimen exposed at the block face is captured. Given the photo position is invariant, the 2D image stack collected is in perfect registration, and thus can be digitally resectioned in any imaging plane. It also can be rapidly reconstructed to generate high-resolution 3D renderings of the specimen. In the first generation EFIC imaging system, surface fluorescence is captured with epifluorescence imaging using standard mercury illumination. To prevent bleed through fluorescence from beneath the block surface, aniline, a light blocking dye is incorporated into the paraffin wax via a laborious and difficult high temperature embedding process. With this proposal, we will assemble a 2nd generation EFIC imaging system that will obviate the need for high temperature paraffin embedding with aniline. Imaging will be carried out with the Leica widefield superzoom LSI confocal macroscope. As confocal imaging will allow capture of only surface fluorescence, it will eliminate the need for the incorporation of aniline dye in the paraffn. This makes it possible for any paraffin embedded specimen to be processed for EFIC imaging. In addition, with appropriate lasers and excitation/emission filters, EFIC imaging can be combined with the tracking of specific cell lineages using fluorescent protein tagged expression constructs. Overall, EFIC imaging achieves better resolution than images obtained by MRI, and has the advantage that it can be learned easily by most investigators, given it is essentially a histological technique combined with confocal imaging. The ability to digitally resection the 2D image stacks in any imaging plane and the ease for rapid 3D reconstructions are invaluable for the assessment and diagnosis of anatomical malformations such as that associated with congenital heart disease, or defects in vascular patterning or branching morphogenesis. In addition, quantitative assessment of anatomical structures can be carried out using the perfectly registered 2D image stacks or 3D reconstructions, such as vessel/tubule dimensions or their branching complexities, or the size of chamber lumen dimensions or tissue thicknesses. This confocal enhanced EFIC system will be assembled using off the shelf equipment, the Leica SM2500 microtome and the Leica LSI confocal macroscope. Customization required to affix and align the confocal macroscope to the photo position of the microtome has already been developed with generous seed funding from the School of Medicine and with the borrowed use of an LSI confocal mascroscope from the Department of Developmental Biology. Therefore, funding requested in this proposal will allow the immediate installation of a dedicated confocal EFIC imaging system. This will greatly enhance the research programs of six well funded NIH investigators, filling a gap in 2D/3D imaging technology for high resolution tissue structure analysis. PUBLIC HEALTH RELEVANCE: This application seeks to acquire a whole tissue imaging system that will provide detailed visualization of anatomical structures in three dimensions. This will facilitate the detection of alterations in tissues architecture associated with birth defects nd other acquired diseases, and allows quantitative analysis of anatomical and tissue defects to yield novel insights into disease mechanisms.