Molecular Imaging of Biomaterials - Single Cells

生物材料的分子成像 - 单细胞

• 批准号: 6999725
• 负责人: NICHOLAS WINOGRAD
• 金额: $ 34.17万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6999725
• 关键词: animal tissue; bioimaging /biomedical imaging; exocytosis; freeze etching; imaging /visualization /scanning; intracellular membranes; intracellular transport; mass spectrometry; mast cell; neurotransmitter transport; neurotransmitters; phospholipids; synaptic vesicles; technology /technique development; tissue /cell culture; tissue /cell preparation

DESCRIPTION (provided by applicant): The long-term goal of this research is to establish the chain of molecular events associated with neurotransmitter release at the single cell and subcellular level. Specifically, the spatial and temporal behavior of small molecules such as dopamine, serotonin, and histamine, and the domain structure of phospholipid membrane bilayers involved in the process of exocytosis will be determined. To establish these structures, a mass spectrometry-based molecule-specific imaging protocol is being developed. This protocol utilizes a specialized freeze-fracture device that allows cells to be quenched in the laboratory and sectioned in a sample preparation chamber of the mass spectrometer. Mass spectra are acquired by utilizing a less than 100 nm-focused energetic ion beam to desorb molecular ions into a time-of flight mass spectrometer. Images are constructed by rastering the ion beam over the target cells and collecting mass spectra at each pixel. There are three aims for this proposal. First, although adequate sensitivity is available to acquire the requisite molecule-specific images, there are emerging possibilities that can significantly boost signal levels, and hence lateral resolution. Plans include implementation of gold and C60 ion sources constructed over the last two years, molecular depth profiling in an ice matrix whereby the number of available molecules for imaging is significantly increased, laser postionization to detect the desorbed neutral molecules, and laser desorption imaging directly from ice using femtosecond UV pulses for mapping protein signals. Second, to provide a basis for cell imaging experiments, there are plans to expand the repertoire of model membrane systems necessary to establish the efficacy of the mass spectrometry experiments. Model systems include Langmuir-Blodgett films doped with cholesterol that form rafts and liposome networks. The networks can be enticed to form domains and act as models for artificial exocytosis. Third, these protocols will be utilized to study the dynamics of membrane chemistry and neurotransmission in single cells. Candidates include the study of histamine release from mast cells, the study of membrane chemistry after vesicle fusion and the assay of neurotransmitter levels in the solution (halo) around the dense core vs. the core of individual vesicles. To test the hypotheses put forth, measurements of vesicles and single events specific to individual cells are required. This scientific agenda will provide valuable information toward understanding the molecular basis of brain-related disease states, which according to recent hypotheses involve lipid rafts. These diseases include Alzheimer's, Parkinson's and a variety of autoimmune conditions.

描述（由申请人提供）：这项研究的长期目标是在单细胞和亚细胞水平上建立与神经递质释放相关的分子事件链。具体而言，将确定小分子（例如多巴胺，5-羟色胺和组胺）的空间和时间行为，以及参与胞外增生过程的磷脂膜双层的结构结构。为了建立这些结构，正在开发基于质谱的分子特异性成像方案。该协议采用了专门的冻结装置，该设备允许在实验室中淬灭细胞并在质谱仪的样品制备室中进行切片。通过利用小于100 nm的能量离子束将分子离子排列到飞行时间质谱仪中，获得质谱。图像是通过将离子束在目标细胞上栅格栅格并在每个像素上收集质谱而构建的。该提案有三个目标。首先，尽管有足够的灵敏度可用于获取必要的分子特异性图像，但有一些新兴的可能性可以显着提高信号水平，从而横向分辨率。计划包括实施过去两年中构建的黄金和C60离子源，分子深度谱图在冰基质中进行分子深度分析，从而显着增加了用于成像的可用分子的数量，激光后置换以检测令人脱附的中性分子，并直接从使用fmtosecond uv pulses的冰蛋白蛋白质蛋白质蛋白质蛋白质蛋白质signers signers signals来检测激光吸收成像。其次，为了提供细胞成像实验的基础，有计划扩展建立质谱实验效果所需的模型膜系统的曲目。模型系统包括langmuir-blodgett膜，掺有胆固醇，形成木筏和脂质体网络。可以吸引网络形成域并充当人工胞吞作用的模型。第三，这些方案将用于研究单细胞中膜化学和神经传递的动力学。候选者包括研究从肥大细胞中释放组胺的研究，囊泡融合后的膜化学研究以及围绕密集核心与单个囊泡核心的溶液中神经递质水平的测定。为了测试提出的假设，需要测量囊泡和特定于单个细胞的单个事件。该科学议程将提供有价值的信息，以了解与脑相关疾病状态的分子基础，根据最近的假设涉及脂质筏。这些疾病包括阿尔茨海默氏症，帕金森氏症和各种自身免疫性疾病。