Confocal enhanced episcopic fluorescent image capture (EFIC)

描述（由申请人提供）：本申请寻求获得共聚焦增强主显荧光图像捕获（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。当每个切片使用切片机与固定的照片位置切割时，暴露在块表面的标本的荧光图像被捕获。由于照片位置不变，采集到的二维图像堆栈是完美配准的，因此可以在任何成像平面上进行数字切割。它还可以快速重建以生成标本的高分辨率3D渲染图。在第一代EFIC成像系统中，表面荧光是用标准汞照明的表观荧光成像捕获的。为了防止从块表面下的荧光渗出，苯胺，一种光阻挡染料通过费力和困难的高温包埋过程掺入石蜡中。根据这一建议，我们将组装第二代EFIC成像系统，该系统将省去高温石蜡包埋苯胺的需要。成像将进行与徕卡广角超变焦LSI共聚焦宏观。由于共聚焦成像只允许捕获表面荧光，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标本都可以进行EFIC成像处理。此外，使用适当的激光和激发/发射滤波器，EFIC成像可以与使用荧光蛋白标记表达构建的特定细胞系的跟踪相结合。总的来说，EFIC成像比MRI获得的图像具有更好的分辨率，并且由于它本质上是一种与共聚焦成像相结合的组织学技术，因此大多数研究人员可以很容易地了解它。在任何成像平面上数字切除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）或血管模式或分支形态发生缺陷是无价的。此外，可以使用完美注册的2D图像堆栈或3D重建进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，或腔腔尺寸或组织厚度的大小。这种共聚焦增强EFIC系统将使用现成的设备，徕卡SM2500显微切片机和徕卡LSI共聚焦显微镜进行组装。在医学院慷慨的种子基金和从发育生物系借来的LSI共聚焦显微镜的帮助下，已经开发出了将共聚焦显微镜固定和对准显微镜照片位置的定制技术。因此，本提案所要求的经费将允许立即安装专用的共聚焦EFIC成像系统。这将极大地加强六位NIH研究人员的研究计划，填补高分辨率组织结构分析的2D/3D成像技术的空白。

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.