Confocal enhanced episcopic fluorescent image capture (EFIC)

共焦增强型落射荧光图像捕获 (EFIC)

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/8246865
• 关键词: Aniline; Blood Vessels; Cell Lineage; Defect; Developmental Biology; Diagnosis; Dimensions; Dyes; Equipment; Excision; Face; Fluorescence; Funding; Generations; Hemorrhage; High temperature of physical object; Histological Techniques; Image; Lasers; Learning; Light; Lighting; Magnetic Resonance Imaging; Mercury; Microtome - medical device; Morphogenesis; Paraffin; Paraffin Embedding; Pattern; Positioning Attribute; Process; Proteins; Research; Research Personnel; Resolution; Seeds; Specimen; Structure; Surface; System; Technology; Thick; Three-Dimensional Imaging; Tissues; United States National Institutes of Health; Waxes; aniline dye; congenital heart disorder; fluorescence imaging; malformation; medical schools; prevent; programs; reconstruction

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(申请人提供)：本申请寻求获得一种共焦增强型表观荧光图像捕获(EFIC)系统。EFIC成像需要在石蜡包埋标本的切面捕获组织荧光。当使用具有固定照片位置的显微切割器切割每个切片时，在块面上曝光的样品的荧光图像被捕获。由于照片的位置是不变的，所以收集的2D图像堆栈处于完美的配准状态，因此可以在任何成像平面上进行数字切割。它还可以被快速重建，以生成高分辨率的样本3D渲染。在第一代EFIC成像系统中，表面荧光是用标准汞照明的荧光成像来捕获的。为了防止通过荧光从块表面下面渗出，通过费力而困难的高温包埋过程，将阻光染料苯胺加入石蜡中。有了这个提议，我们将组装第二代EFIC成像系统，它将消除高温石蜡包埋苯胺的需要。成像将使用徕卡广域超变焦LSI共焦显微镜进行。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的标本都可以被处理以进行EFIC成像。此外，通过适当的激光和激发/发射过滤器，EFIC成像可以与使用荧光蛋白标记表达构建体追踪特定细胞谱系相结合。总体而言，EFIC成像获得了比MRI获得的图像更好的分辨率，并且具有大多数研究人员可以很容易地学习的优势，因为它本质上是一种组织学技术与共聚焦成像相结合。数字切除任何成像平面上的2D图像堆叠的能力和快速3D重建的简便性对于评估和诊断解剖畸形，如与先天性心脏病相关的畸形，或血管构型或分支形态发生方面的缺陷，是非常宝贵的。此外，可以使用完美配准的2D图像堆叠或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，或腔腔尺寸或组织厚度的大小。这种共焦增强型EFIC系统将使用现成的设备、徕卡SM2500显微镜和徕卡LSI共焦显微镜进行组装。在医学院慷慨的种子资金和从发育生物学系借用的LSI共焦显微镜的使用下，已经开发出将共焦显微镜固定并对准显微切片机的照片位置所需的定制。因此，本提案中要求的资金将允许立即安装一个专用的共焦EFIC成像系统。这将极大地加强六名资金充足的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。 公共卫生相关性：该应用程序寻求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。

项目成果

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.

• DOI: 10.1002/bdr2.1048
• 发表时间: 2017-06-01
• 期刊: Birth defects research
• 影响因子: 2.1
• 作者: Liu X;Kim AJ;Reynolds W;Wu Y;Lo CW
• 通讯作者: Lo CW