MOLECULAR IMAGING OF BIOMATERIALS - SINGLE CELLS

生物材料的分子成像 - 单细胞

基本信息

批准号：
6490970
负责人：
NICHOLAS WINOGRAD
金额：
$ 32.32万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-30 至 2003-12-31
项目状态：
已结题

项目摘要

Our goal is to develop and apply mass spectrometric molecular imaging to examine phospholipid domain structure and spatial patterns of neurotransmitter release at single cells. This will be accomplished by use of mass spectrometry as a novel tool for acquiring molecule-specific images of single cells with subcellular spatial resolution. The specific focus of this proposal is to develop the necessary protocols to examine both neurotransmitters and membrane phospholipid chemistry at the subcellular level with a special emphasis on unraveling the mechanism of exocytosis. The instrumentation utilizes a liquid metal ion source which is focused to a size of 50 or 200 nanometers. Molecules are desorbed from this small area into a time-of-flight mas spectrometer. Images are recorded by scanning the beam across the target and acquiring mass spectra at each pixel. Our groups have recently demonstrated the capability of detecting several small molecules directly from single cells. The key to performing these measurements has been development of a special freeze-fracture protocol that allows a cell to be frozen in the laboratory and sectioned in the vacuum environment of the mass spectrometer. Three parallel strategies for implementation of our goal are proposed. First, we plan to continue efforts to enhance the information content associated with the mass spectra. These plans included expanded options for freeze fracture, laser positionization experiments to detect the desorbed neutral species, and the development of a unique cluster ion beam probe which would enhance molecular ion yields by a factor of 10 or more. Second, we plan to construct model systems consisting of Langmuir-Blodgett and liposome phospholipid layers that will define mass spectral parameters for characterization of the structure and dynamics of membrane chemistry. Preliminary data suggest that it is feasible to characterize domains of specific phospholipid molecules, to assess their molecular orientation and to monitor dynamic behavior all at the micrometer spatial dimension. Finally, we plan to develop aq series of experiments encompassing the above protocols and models to elucidate the molecular aspects that take place in and at the cellular membrane following exocytosis. Specifically, we propose to image phospholipids in subcellular domains of the cell and the spatial distribution of neurotransmitters. released during exocytosis at single cells. This work will provide the chemical basis for models of exocytosis (vesicle fusion with the cell membrane) and endocytosis (vesicle recycling).

我们的目标是开发和应用质谱分子成像，以检查单个细胞在神经递质释放的磷脂结构结构和空间模式。这将通过使用质谱法作为获取具有亚细胞空间分辨率的单个细胞的分子特异性图像的新工具来完成。该提案的具体重点是开发必要的方案，以检查亚细胞水平的神经递质和膜磷脂化学，并特别强调揭示胞吞作用的机制。该仪器利用液态金属离子源，该液体聚焦为50或200纳米。分子从这个小区域分为飞行时间的MAS光谱仪。图像是通过扫描横梁跨目标并在每个像素上获取质谱的记录。我们的小组最近证明了直接从单个细胞中检测几个小分子的能力。执行这些测量值的关键是开发了一种特殊的冻结裂纹方案，该方案允许在实验室中冷冻细胞并在质谱仪的真空环境中进行切片。提出了三种实施我们目标的平行策略。首先，我们计划继续努力增强与质谱相关的信息内容。这些计划包括扩大冻结裂缝的选择，激光定位实验以检测解吸的中性物种，以及独特的簇离子束探针的发展，该探针将增强分子离子的产量10倍或更多。其次，我们计划构建由Langmuir-Blodgett和脂质体磷脂层组成的模型系统，这些系统将定义质谱参数，以表征膜化学的结构和动力学。初步数据表明，表征特定磷脂分子的结构域，评估其分子取向并监测千分尺空间尺寸的动态行为是可行的。最后，我们计划开发一系列AQ系列实验，其中包含上述方案和模型，以阐明胞吐作用后细胞膜和在细胞膜上发生的分子方面。具体而言，我们建议在细胞的亚细胞结构域和神经递质的空间分布中成像磷脂。在单细胞的胞吐作用期间释放。这项工作将为胞吐作用模型（与细胞膜融合）和内吞作用（囊泡回收）提供化学基础。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

C60 secondary ion mass spectrometry with a hybrid-quadrupole orthogonal time-of-flight mass spectrometer.

DOI：
10.1021/ac801712s
发表时间：
2008-11-01
期刊：
ANALYTICAL CHEMISTRY
影响因子：
7.4
作者：
Carado, Anthony;Passarelli, M. K.;Kozole, Joseph;Wingate, J. E.;Winograd, Nicholas;Loboda, A. V.
通讯作者：
Loboda, A. V.

C(60)-SIMS Studies of Glycerophospholipid in a LIPID MAPS Model System: KDO(2)-Lipid A Stimulated RAW 264.7 Cells.