Confocal enhanced episcopic fluorescent image capture (EFIC)

共焦增强型落射荧光图像捕获 (EFIC)

基本信息

批准号：
8246865
负责人：
CECILIA W. LO
金额：
$ 36.83万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2013-04-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：本申请旨在获得共同的增强的上场荧光图像捕获（EFIC）系统。 EFIC成像需要在石蜡嵌入标本的切割表面捕获组织荧光。当每个截面使用具有固定照片位置的微型集体切割时，捕获了暴露在块面上的样品的落荧光图像。鉴于照片位置是不变的，因此收集的2D图像堆栈是完美的注册，因此可以在任何成像平面中进行数字切除。它也可以迅速重建以生成样品的高分辨率3D渲染。在第一代EFIC成像系统中，使用标准汞照明捕获了表面荧光。为了防止通过荧光从砌块表面下方流出，苯胺，通过费力且困难的高温嵌入过程将光阻断染料掺入石蜡蜡中。通过此提案，我们将组装一个第二代EFIC成像系统，该系统将消除对用苯胺嵌入高温石蜡的需求。成像将使用Leica Wideffield SuperZoom LSI共聚焦宏观镜进行。由于共聚焦成像将仅捕获表面荧光，因此它将消除在石膏中掺入苯胺染料的需求。这使得任何石蜡嵌入的试样都可以用于EFIC成像。此外，使用适当的激光和激发/发射过滤器，可以使用荧光蛋白标记的表达构建体的特定细胞谱系结合EFIC成像。总体而言，EFIC成像的分辨率比MRI获得的图像更好，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结合共聚焦成像的组织学技术。在任何成像平面中的2D图像堆叠以及快速3D重建的易于构建的能力对于评估和诊断解剖畸形，例如与先天性心脏病或血管构成图案缺陷或血管构图或分支形成形成相关的解剖畸形，这是无价的。此外，可以使用完美的注册的2D图像堆栈或3D重建，例如血管/小管尺寸或其分支复杂性，或室内流明尺寸或组织厚度的大小来对解剖结构进行定量评估。该共聚焦增强的EFIC系统将使用架子设备，Leica SM2500微型摩托组和Leica LSI共聚焦宏观镜组装。将共焦宏观镜贴在微型群的照片位置所需的自定义已经通过医学院的慷慨种子资金开发，并且通过借用了发育生物学系的LSI共焦大麻镜。因此，本提案中要求的资金将允许立即安装专用的共聚焦EFIC成像系统。这将大大增强六位资助的NIH研究者的研究计划，以填补用于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：该应用程序旨在获取整个组织成像系统，该系统将在三个维度上提供解剖结构的详细可视化。这将有助于检测与先天缺陷和其他获得性疾病相关的组织结构的改变，并允许对解剖学和组织缺陷进行定量分析，以产生对疾病机制的新见解。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Diverse application of MRI for mouse phenotyping.

MRI 在小鼠表型分析中的多样化应用。

DOI：
10.1002/bdr2.1051
发表时间：
2017
期刊：
Birth defects research
影响因子：
2.1
作者：
Wu,YijenL;Lo,CeciliaW
通讯作者：
Lo,CeciliaW

MMP21 is mutated in human heterotaxy and is required for normal left-right asymmetry in vertebrates.

DOI：
10.1038/ng.3376
发表时间：
2015-11
期刊：
Nature genetics
影响因子：
30.8
作者：
Guimier A;Gabriel GC;Bajolle F;Tsang M;Liu H;Noll A;Schwartz M;El Malti R;Smith LD;Klena NT;Jimenez G;Miller NA;Oufadem M;Moreau de Bellaing A;Yagi H;Saunders CJ;Baker CN;Di Filippo S;Peterson KA;Thiffault I;Bole-Feysot C;Cooley LD;Farrow EG;Masson C;Schoen P;Deleuze JF;Nitschké P;Lyonnet S;de Pontual L;Murray SA;Bonnet D;Kingsmore SF;Amiel J;Bouvagnet P;Lo CW;Gordon CT
通讯作者：
Gordon CT

Phenotyping cardiac and structural birth defects in fetal and newborn mice.