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）：本申请旨在获得共焦增强落射荧光图像捕获（EFIC）系统。 EFIC 成像需要捕获石蜡包埋样本切割表面的组织荧光。当使用具有固定照片位置的切片机切割每个切片时，捕获在块面暴露的样本的落射荧光图像。鉴于照片位置不变，收集的 2D 图像堆栈完美配准，因此可以在任何成像平面上进行数字切除。它还可以快速重建以生成样本的高分辨率 3D 渲染图。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。为了防止块表面下方的荧光渗透，苯胺（一种遮光染料）通过费力且困难的高温嵌入过程掺入石蜡中。根据此提案，我们将组装第二代 EFIC 成像系统，该系统将消除使用苯胺进行高温石蜡包埋的需要。将使用徕卡宽视场超变焦 LSI 共焦宏观显微镜进行成像。由于共焦成像只能捕获表面荧光，因此无需在石蜡中加入苯胺染料。这使得任何石蜡包埋标本都可以进行 EFIC 成像处理。此外，通过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表达构建体跟踪特定细胞谱系相结合。总体而言，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点，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化切除 2D 图像堆栈的能力以及快速 3D 重建的便利性对于评估和诊断解剖畸形（例如与先天性心脏病相关的畸形或血管图案或分支形态发生的缺陷）非常有价值。此外，可以使用完美配准的 2D 图像堆栈或 3D 重建来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，或腔室管腔尺寸或组织厚度的大小。该共焦增强型 EFIC 系统将使用现成设备、Leica SM2500 切片机和 Leica LSI 共焦宏观显微镜进行组装。在医学院的慷慨种子资金和发育生物学系借用的 LSI 共焦宏观镜的帮助下，已经开发出了将共焦宏观显微镜固定并对准切片机照片位置所需的定制功能。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 研究人员的研究项目，填补高分辨率组织结构分析 2D/3D 成像技术的空白。公共健康相关性：该应用程序旨在获得一个完整的组织成像系统，该系统将提供三维解剖结构的详细可视化。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的改变，并允许对解剖和组织缺陷进行定量分析，从而对疾病机制产生新的见解。

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）

Role of cilia in structural birth defects: insights from ciliopathy mutant mouse models.

纤毛在结构性出生缺陷中的作用：来自纤毛病突变小鼠模型的见解。

DOI：
10.1002/bdrc.21067
发表时间：
2014-06-01
期刊：
Birth defects research. Part C, Embryo today : reviews
影响因子：
0
作者：
Rama Rao Damerla;George C Gabriel;You Li;N. Klena;Xiaoqin Liu;Yu Chen;Cheng Cui;G. Pazour;C. Lo
通讯作者：
C. Lo

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

MMP21 在人类异位性中发生突变，是脊椎动物正常左右不对称所必需的。

DOI：
发表时间：
2015-11
期刊：
影响因子：
30.8
作者：
Guimier, Anne;Gabriel, George C;Bajolle, Fanny;Tsang, Michael;Liu, Hui;Noll, Aaron;Schwartz, Molly;El Malti, Rajae;Smith, Laurie D;Klena, Nikolai T;Jimenez, Gina;Miller, Neil A;Oufadem, Myriam;Moreau de Bellaing, Anne;Yagi, Hisato;Saunders
通讯作者：
Saunders

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

对胎儿和新生小鼠的心脏和结构性出生缺陷进行表型分析。