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：
8231477
负责人：
NICHOLAS WINOGRAD
金额：
$ 41.18万
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 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8231477
关键词：
3-Dimensional Achievement Alzheimer&apos s Disease Area Autoimmune Diseases Autoimmune Process Base of the Brain Behavior Biocompatible Materials Biological Biological Assay Biological Models Biophysics Caliber Cell Communication Cell membrane Cell physiology Cells Chemicals Chemistry Cholesterol Communities Detection Development Devices Disease Dopamine Effectiveness Equipment Event Exocytosis Film Fluorescence Microscopy Fracture Freeze Fracturing Freezing Funding Goals Grant Health Histamine Histamine Release Image In Situ Individual Ions Knowledge Laboratories Lasers Lateral Lipids Liposomes Mass Spectrum Analysis Measurement Measures Membrane Methods Modality Modeling Molecular Neurotransmitters PC12 Cells Parkinson Disease Partner in relationship Pathway interactions Pattern Phospholipids Play Preparation Process Property Proteins Protocols documentation Protons Research Resolution Resources Role Sampling Site Solutions Source Specificity Specimen Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Spectrometry, Mass, Secondary Ion Spottings Structure Surface Synaptic Membranes System Technology Testing Tetrahymena Time Tissues United States National Institutes of Health Vesicle Work base biological research cellular imaging design efficacy testing improved instrument instrumentation interest ionization mass analyzer mass spectrometer mast cell membrane model molecular imaging nanoscale neurotransmission neurotransmitter release novel particle public health research research study small molecule submicron success theories tool

项目摘要

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. This goal includes the characterization of the spatial, and domain structure of phospholipid membrane layers and the temporal behavior of small molecules such as dopamine, seratonin, and histamine involved in the process of exocytosis. The central approach will be to utilize an advanced bioanalytical mass spectrometry-based protocol to acquire two and three dimensional molecule-specific image information at the nanoscale level. The instrumentation involves using a specialized freeze-fracture device which 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 an energetic beam comprised of molecular cluster ions focused to a sub-micron spot diameter directly onto single cells. Molecular ions are desorbed into a time-of flight mass spectrometer with images constructed by rastering the ion beam over the field of view and collecting mass spectra at each pixel. There are four specific aims for this proposal. First, although adequate sensitivity is available to acquire the requisite molecule-specific images, there are opportunities to build on the capabilities of the instrumentation even further, given recent success in implementation of Au3, Bi3 and C60 cluster beams. Plans include enhancement of secondary ion yields by optimizing the special properties of cluster beams to produce protons in the near surface region of the specimen, molecular depth profiling, possible for the first time with cluster beams where chemical damage buildup is mitigated, 3- dimensional measurements whereby the number of available molecules for imaging is significantly increased, laser post- ionization to detect the desorbed neutral molecules, and implementation of new tandem MS instrumentation that more effectively optimizes the properties of the new cluster sources. 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. These model systems include Langmuir-Blodgett films-and liposome spheres doped with varying lipid/protein/small-molecule combinations that can be enticed to form domains and to 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 dense core vs. the core of individual vesicles. These experiments require measurements at single cells, and off single events or vesicles to test the hypotheses put forth. Fourth, a new instrument utilizing an orthogonal TOF geometry with tandem MS will be employed for SIMS imaging at higher mass resolution, and better sensitivity to events that are occurring near the synaptic membrane. This scientific agenda will provide valuable information toward understanding the molecular basis of brain-related disease states such as Alzheimer's and Parkinson's disease and a variety of autoimmune conditions recently hypothesized to involve lipid domains. PUBLIC HEALTH RELEVANCE: We propose to continue to develop an exciting area of mass spectrometric imaging and to apply this method to understand the structure and biophysics of cell membranes in regulating neurotransmitter exocytosis. In addition, cholesterol will be probed as a marker of lipid domains that might play a role in determining, for example, Alzheimer's and autoimmune disease states. A major objective is to establish the unique imaging strategy as a valuable new tool for use by the larger biological and public health research community.

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。该目标包括表征磷脂膜层的空间和结构域结构，以及参与胞吐作用过程中的多巴胺，血清素和组胺等小分子的时间行为。中心方法是利用基于先进的生物分析质谱法的方案在纳米级级别获取两个和三维分子特异性图像信息。该仪器涉及使用专门的冻结装置，该装置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。质谱是通过利用由分子簇离子组成的能量束来获取的，该分子簇离子聚焦于直径直接在单个细胞上。分子离子被吸引到飞行时间质谱仪中，图像是通过在视野上栅格栅格构建的图像，并在每个像素上收集质谱。该提案有四个具体的目标。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，但鉴于最近在实施AU3，BI3和C60群集光束方面取得了成功，因此有机会进一步建立仪器的能力。 Plans include enhancement of secondary ion yields by optimizing the special properties of cluster beams to produce protons in the near surface region of the specimen, molecular depth profiling, possible for the first time with cluster beams where chemical damage buildup is mitigated, 3- dimensional measurements whereby the number of available molecules for imaging is significantly increased, laser post- ionization to detect the desorbed neutral molecules, and实施新的串联MS仪器，更有效地优化了新集群源的属性。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。这些模型系统包括langmuir-blodgett膜和脂质体球，掺有不同的脂质/蛋白质/小分子组合，可以吸引形成域并充当人工胞吐的模型。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在致密核心与单个囊泡的核心周围溶液中（HALO）中神经递质水平的测定。这些实验需要在单个细胞上进行测量，并在单个事件或囊泡上进行测试以测试提出的假设。第四，将使用带有串联MS的正交TOF几何形状的新仪器用于更高质量分辨率的SIMS成像，并且对突触膜附近发生的事件的敏感性更好。该科学议程将提供有价值的信息，以了解与大脑相关疾病状态的分子基础，例如阿尔茨海默氏症和帕金森氏病，以及最近假设的各种自身免疫性疾病，涉及脂质域。公共卫生相关性：我们建议继续开发一个令人兴奋的质谱成像领域，并应用这种方法来了解调节神经递质胞吐作用时细胞膜的结构和生物物理学。此外，胆固醇将被探测为脂质结构域的标志物，这些脂质结构域可能在确定例如阿尔茨海默氏病和自身免疫性疾病状态下发挥作用。一个主要目标是建立独特的成像策略，作为较大的生物学和公共卫生研究社区使用的宝贵新工具。

项目成果

期刊论文数量（72）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Fluid Flow and Effusive Desorption: Dominant Mechanisms of Energy Dissipation after Energetic Cluster Bombardment of Molecular Solids.

DOI：
10.1021/jz200708j
发表时间：
2011-07-22
期刊：
The journal of physical chemistry letters
影响因子：
0
作者：
Brenes DA;Postawa Z;Wucher A;Blenkinsopp P;Garrison BJ;Winograd N
通讯作者：
Winograd N

Mass spectral imaging of glycophospholipids, cholesterol, and glycophorin a in model cell membranes.

DOI：
10.1021/la802582f
发表时间：
2008-10-21
期刊：
LANGMUIR
影响因子：
3.9
作者：
Baker, Matthew J.;Zheng, Leiliang;Winograd, Nicholas;Lockyer, Nicholas P.;Vickerman, John C.
通讯作者：
Vickerman, John C.

Improving secondary ion mass spectrometry image quality with image fusion.

通过图像融合提高二次离子质谱图像质量。