ShEEP Request for Laser Microdissection Instrument

ShEEP 请求激光显微切割仪器

• 批准号: 9796446
• 负责人: WILLARD M FREEMAN
• 金额: --
• 依托单位: OKLAHOMA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2019-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9796446
• 关键词: Aging; Antibodies; Archives; Award; Biochemical; Cell Culture Techniques; Cell Separation; Cell physiology; Cell surface; Cells; Cellular Structures; Cities; Collecting Cell; Collection; Computer software; Computers; DNA Methylation; Disease; Dissociation; Doctor of Philosophy; Ensure; Freezing; Fresh Tissue; Functional disorder; Funding; Future; Gene Expression; Genomics; Grant; Hand; Health; Health Sciences; Heterogeneity; Image; K-Series Research Career Programs; Lasers; Maintenance; Malignant Neoplasms; Medical center; Microdissection; Microscope; Microscopic; Mitochondria; Molecular; Molecular Analysis; Neurosciences; Neurosciences Research; Oklahoma; Organ; Process; Productivity; Reporter; Research; Research Personnel; Resolution; Robotics; Route; Sampling; Scientist; Shapes; Sheep; Shock; Sorting - Cell Movement; Specificity; Stains; Structure; Surveys; System; Techniques; Technology; Tissue Sample; Tissues; Training; Transgenic Organisms; Universities; Work; anticancer research; base; career; cell type; epigenomics; immunohistochemical markers; improved; insight; instrument; instrumentation; interest; laser capture microdissection; programs; research facility; sample collection; transcriptomics

We are requesting funds to purchase a Zeiss PALM Microbeam laser capture microdissections system. This instrument allows investigators to dissect and collect, down to the single cell level, specific fields, structures, and cells from tissue sections. Currently, neither the research facility at the Oklahoma City VA Medical Center (OKC VAMC) or the affiliated University of Oklahoma Health Sciences Center have the ability to perform this type of microscopic sample collection. It is well known that a principle limitation to analyses of bulk tissue is that there is a heterogeneity of cell types and any resultant molecular or biochemical analyses reflect the cellular status across this mixture of cells. Being able to analyze each cell type or structure separately has great advantages for understanding cellular function in health and disease and how different changes across multiple cell types in a tissue work in a cell non-autonomous fashion to cause tissue dysfunction and disease. Traditionally, the approach to resolving this challenge is to use cell sorting techniques. While cell sorting approaches are invaluable, in many cases they must be performed on fresh tissue, have appropriate cell surface markers or transgenic reporters, and require extensive technical optimization to ensure that cells remain unchanged during this process. An alternative approach is to microscopically cut specific cells out of tissue sections. In this approach tissues are collected in an intact form, either fresh frozen or fixed, sectioned and then imaged on the laser capture microscope. The cells or structures of interest and then excised with a computer controlled laser. These cells can then be used for a wide variety molecular and biochemical analyses. The only requirement is that there be some form of structure, stain, or immunohistochemical marker to identify the cell type(s) of interest. With this instrument the microdissected samples then are collected in a contactless manner ensuring sample purity and ability to use for many types of downstream molecular and biochemical analyses. The Zeiss PAM Microbeam instrument was selected based on the versatility of the instrument to meet the various needs of OKC VAMC investigators. The ability of this system to collect fields/cell layers is important to neuroscience and cancer investigators wanting to analyze microscopic structures within organs that cannot be dissected by hand. All of the investigators can make use of the ability to detect specific cell types with stains or antibodies and then collect only these cells. A future potential use is the ability to couple cell cultures with this instrument and collect specific types of lives cells for sub-culturing. Also central to instrument selection was the ability to ‘teach’ the robotic software to recognize a specific cell types based on the staining, size and shape to collect cell automatically. A survey of all VA researchers at OKC VAMC showed a wide variety of researcher will utilize the system. This instrument will be used by five major users and a number of future users who have six VA Merit grants funded, three senior career scientist awards, and one career development award funded.

我们正在申请资金购买 Zeiss PALM Microbeam 激光捕获显微切割系统。 该仪器允许研究人员解剖和收集，直至单细胞水平、特定区域、 结构和组织切片的细胞。目前，俄克拉荷马城弗吉尼亚州的研究机构 医学中心（OKC VAMC）或附属的俄克拉荷马大学健康科学中心有能力 执行这种类型的显微样品采集。众所周知，分析的原则限制 大块组织的特点是细胞类型和任何由此产生的分子或生化分析存在异质性 反映了该细胞混合物中的细胞状态。能够分析每种细胞类型或结构 分别对于了解健康和疾病中的细胞功能以及有何不同具有巨大的优势 组织中多种细胞类型的变化以细胞非自主方式起作用，导致组织 功能障碍和疾病。传统上，解决这一挑战的方法是使用细胞分选 技术。虽然细胞分选方法非常有价值，但在许多情况下，它们必须在 新鲜组织，具有适当的细胞表面标记或转基因报告基因，并且需要广泛的技术 优化以确保细胞在此过程中保持不变。另一种方法是 显微镜下从组织切片中切下特定细胞。在这种方法中，组织被完整地收集 形式，新鲜冷冻或固定，切片，然后在激光捕获显微镜上成像。细胞或 感兴趣的结构，然后用计算机控制的激光切除。然后这些细胞可以用于 各种各样的分子和生化分析。唯一的要求是有某种形式的 结构、染色或免疫组织化学标记来识别感兴趣的细胞类型。有了这个仪器 然后以非接触方式收集显微解剖样品，确保样品纯度和使用能力 适用于多种类型的下游分子和生化分析。 选择蔡司PAM Microbeam仪器是基于仪器的多功能性，可以满足各种 OKC VAMC 调查员的需求。该系统收集场/细胞层的能力对于 神经科学和癌症研究人员想要分析器官内无法进行的微观结构 用手解剖。所有研究人员都可以利用通过染色或检测特定细胞类型的能力 抗体，然后仅收集这些细胞。未来的潜在用途是将细胞培养物与此结合的能力 仪器并收集特定类型的生命细胞进行传代培养。仪器选择的另一个核心是 能够“教导”机器人软件根据染色、大小和形状识别特定细胞类型 自动收集细胞。 对 OKC VAMC 的所有 VA 研究人员的调查显示，各种各样的研究人员都会使用该系统。这 该仪器将由五个主要用户和一些拥有六项 VA 优异补助金的未来用户使用， 资助了三项高级职业科学家奖和一项职业发展奖。