Sample prep methods to allow automated 3D analysis of microvessel morphology

样品制备方法可对微血管形态进行自动 3D 分析

• 批准号: 7413320
• 负责人: Rick Rogers
• 金额: $ 22.12万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7413320
• 关键词: Address; Angiogenesis Inhibitors; Animal Model; Animals; Antibodies; Architecture; Archives; Behavior; Biological Models; Biology; Biopsy; Biopsy Specimen; Blood Vessels; Blood capillaries; Cancerous; Cardiac; Characteristics; Collection; Computer Analysis; Computer software; Confocal Microscopy; Contrast Media; Corrosion Casting; Count; Data; Data Quality; Depth; Development; Developmental Biology; Diffusion; Disease Progression; Disseminated Malignant Neoplasm; Distant; Dyes; Evaluation; Figs - dietary; Gelatin; Goals; Green Fluorescent Proteins; Growth and Development function; Human; Image; Image Analysis; Imagery; Individual; Invasive; Investigation; Label; Lectin; Lewis Lung Carcinoma; Life; Liposomes; Magnetic Resonance Imaging; Maintenance; Malignant Neoplasms; Manuals; Methods; Metric; Microtomy; Monitor; Morphology; Mus; None or Not Applicable; Nuclear; Optics; Oxygen; Paraffin; Perfusion; Pliability; Preparation; Principal Investigator; Procedures; Process; Property; Protocols documentation; Range; Research Personnel; Resolution; Rodent; Running; Sampling; Scanning Electron Microscopy; Sepharose; Site; Specimen; Structure; Techniques; Texas red; Three-Dimensional Image; Three-Dimensional Imaging; Three-dimensional analysis; Time; Tissue Harvesting; Tissue Sample; Tissues; Tracer; Tumor Tissue; Vascular Endothelium; Video Microscopy; angiogenesis; anticancer research; base; cancer cell; capillary; capillary bed; comparative; density; human tissue; in vivo; innovation; interest; light microscopy; light scattering; neoplastic cell; programs; reconstruction; response; size; success; tumor; tumor growth; usability

DESCRIPTION (provided by applicant): Abnormalities of vascular morphology resulting from angiogenesis, the formation of new blood vessels, are characteristic of cancerous tumors and are associated with a switch from quiescent to aggressively invasive, metastatic behavior. Thus angiogenic activity, as reflected by morphological microvascular change, is a critical point of assessment in cancer research. Visualization and quantification of microvascular attributes permit monitoring of disease progression and response to therapy, yet there are no currently available sample preparation methods to produce data that are of capillary-resolution and suitable for automated 3D quantitative analysis. Specimen preparation techniques for vascular visualization thus far have primarily been developed to meet the requirements of individual studies and have not addressed the need for standardized quantitative analysis across labs. Routine vascular metrics, such as density, often still rely upon time-consuming manual counting from 2D paraffin sections by multiple observers, in part because of the lack of 3D image data of sufficient quality to allow automated quantification. Moreover, one of the principal techniques allowing the collection of 3D quantitative vascular data, corrosion casting, destroys surrounding tissue, eliminating the possibility of simultaneous probing for molecules of interest. Our goal is to create and refine specimen preparation techniques that allow collection of high-resolution 3D vascular image data within tumors and the supporting peri-tumoral tissue, while simultaneously allowing labeling of related molecules of interest. The methods will be optimized to produce vascular image data that will allow automated quantitative analysis, thus introducing a more rapid, standardized approach for studies of microvascular changes associated with tumor growth and treatment. Methods appropriate to animal models and to fixed archived tissue will be developed toward the ultimate goal of extending these techniques to human biopsy tissue. Our methods will combine and optimize protocols used in vascular biology and adapt procedures developed in other fields, such as non-mammalian developmental biology, that though relevant, have not previously been employed in this realm. We propose to modify these existing specimen preparation techniques, and in concert with optical clearing methods to minimize problems associated with tissue opacity, produce 3D renderings of the microvascular architecture of whole-mount specimens, while permitting labeling of other molecules of interest. Our preparation techniques will employ combinations of: 1) casting of the vasculature by filling with a contrast agent; 2) marking of functional blood vessels by fluorescently labeled lectin administered i.v.; 3) marking of other structures of interest by whole-mount labeling with antibodies or nuclear dyes; and 4) tissue clearing to minimize light scattering for imaging by confocal microscopy so that data can be collected from deep within peri-tumoral and viable tumoral tissue.

描述（由申请人提供）：由血管生成（新血管的形成）引起的血管形态异常是癌性肿瘤的特征，并且与从静止到侵袭性转移行为的转换有关。因此，微血管形态变化所反映的血管生成活性是癌症研究中评估的一个关键点。微血管属性的可视化和量化可以监测疾病进展和对治疗的反应，但目前还没有可用的样品制备方法来产生毛细血管分辨率和适合自动3D定量分析的数据。到目前为止，血管可视化的标本制备技术主要是为了满足个体研究的要求而开发的，并没有解决跨实验室标准化定量分析的需要。常规血管指标，如密度，通常仍然依赖于由多个观察者对二维石蜡切片进行耗时的人工计数，部分原因是缺乏足够质量的三维图像数据来实现自动化定量。此外，主要技术之一允许收集三维定量血管数据，腐蚀铸造，破坏周围组织，消除了同时探测感兴趣分子的可能性。我们的目标是创建和完善标本制备技术，以便收集肿瘤和支持肿瘤周围组织内的高分辨率3D血管图像数据，同时允许标记相关感兴趣的分子。这些方法将被优化，以产生血管图像数据，从而允许自动定量分析，从而引入一种更快速、标准化的方法来研究与肿瘤生长和治疗相关的微血管变化。适合动物模型和固定存档组织的方法将朝着将这些技术扩展到人体活检组织的最终目标发展。我们的方法将结合和优化血管生物学中使用的协议，并适应在其他领域开发的程序，例如非哺乳动物发育生物学，尽管相关，但以前从未在该领域使用过。我们建议修改这些现有的标本制备技术，并与光学清除方法相结合，以最大限度地减少与组织不透明度相关的问题，在允许标记其他感兴趣分子的同时，生成整个标本微血管结构的3D渲染图。我们的制备技术将采用以下几种方法的组合：1)用造影剂填充血管；2)通过静脉注射荧光标记凝集素来标记功能血管；3)用抗体或核染料全贴装标记其他感兴趣的结构；4)组织清理，以减少共聚焦显微镜成像的光散射，从而可以从肿瘤周围和活的肿瘤组织深处收集数据。