Methodology development to map and quantify imaged molecules in brain tissue

绘制和量化脑组织中成像分子的方法学开发

• 批准号: 9552575
• 负责人: Amina Woods
• 金额: $ 40.43万
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9552575
• 关键词: Alpha Particles; Animal Disease Models; Area; Biological Process; Brain; Buffers; Caliber; Cells; Ceramides; Complex; Coupled; Data; Deposition; Detection; Development; Diabetes Mellitus; Diet; Functional disorder; Gold; Hand; Humidity; Image; Implant; Implantation procedure; Ions; Kidney; Lipids; Liquid substance; Maps; Mass Spectrum Analysis; Measurement; Metals; Methodology; Methods; Neurodegenerative Disorders; Obesity; Optics; Organ; Organelles; Phosphatidylethanolamine; Physical condensation; Process; Property; Reproducibility; Role; Sampling; Signal Transduction; Silver; Solvents; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Standardization; Surface; Suspensions; Techniques; Temperature; Time; Tissue imaging; Tissues; Triglycerides; Water; Work; biological systems; brain tissue; cell type; experience; heart imaging; implantation; improved; ion source; ionization; metallicity; migration; millimeter; molecular imaging; monolayer; nanometer; nanoparticle; particle; particle beam; stressor; tool

Previously, we used gold nanoparticles suspended in a buffer as a matrix. However, metallic particles are not stable in suspension making homogeneous deposition challenging. An alternative deposition technique is the implantation of nanoparticles. Implantation is a dry and uniform deposition technique, which initially employed a massive cluster ion source. The gold cluster ion currents were insufficient for implanting large areas, and therefore not suited for full organ imaging. The development of an NPlanter(Ionwerks, Houston, TX) has allowed for whole organ tissue sections to be implanted with metal nanoparticles. In these studies, a silver target is inserted into a particle source NanoGen50. These nanoparticles (NP) are generated by expulsion from the metals surface using magnetron sputtering, and then allowed to grow by condensation in a refinement zone (particles are 0.5-15 nm in diameter). The size is selected with a quadrupole mass filter coupled to the magnetron, so that we can insure that the NP size is reproducible. Presently we are using AgNP 6-7 nm in diameter, so we always have the same size nanoparticles implanted in different tissue sections, making implantation reproducible. Finally, the particle beam can be deviated with electro-optics over a minimum of 400 square millimeters (20 x 20), allowing implantation of a whole tissue section at once. We use the equivalent of 2-4 monolayers. It takes 18 minutes to implant each tissue section. By using the same size NP particles and the same number of monolayers. We have standardized the implantation procedure and made it reproducible. Thus making comparison of tissue implanted at various time possible, as they are more likely to give reproducible data. In addition a tissue section can be reimplanted for in-depth profiling. So far we have been able to reimplant and reimage a tissue section nine times in a row. Each image is of an area 10-15 nanometer deeper. However we still are in the process of confirming the depth. We also were able to image the same tissue section five times in a row after a single implantation. The implanter reduces the inherent variability between operators and reduces the influence of humidity and temperature fluctuations, which makes it consistent across time, samples, and operators. Silver implantation is a dry method, and therefore does not cause any blurring or analyte migration which may occur with solvent dissolved matrix. We found that hand spraying with an artistic airbrush is a fairly reproducible method for depositing organic matrices with minimal lipid disruption. However it can be a work intensive and time consuming process that requires an experienced hand. While in implantation, the rapid automated rastering step achieves excellent uniformity in matrix implantation by precisely covering the whole implanted areas. Total control of the amount of particles implanted at each point also contributes to the improved uniformity and reproducibility of the implantation. The quantity of silver and quality of coverage can be validated through measurement. We also imaged the heart and kidney to demonstrate the reproducibility in all tissue not just brain. This type of imaging is very useful for studying animal models of disease in many organs. Additional studies have been conducted to the type of lipids that favor ionization using AgNPs. Our work has shown AgNPs are an excellent matrix for the ionization of neutral lipids such as ceramides (Cer), galactoseceramides (GalCer), diacyglycerols (DAG), and triacylglycerols (TAG) in positive ion mode. Mass spectrometry imaging (MSI) of Cer and DAG in the past have used the M-water mass peak which is problematic since this mass peak can be produced by other complex lipids. Using AgNPs has enabled MALDI images that map only the intact Cer and DAG and thus no interfering lipid species. In negative ion mode, AgNPs yield results similar to organic matrices but with the added benefit of being a dry coating method.

以前，我们使用了悬浮在缓冲液中的金纳米颗粒作为基质。但是，金属颗粒在悬浮液中不稳定，从而使均匀的沉积具有挑战性。另一种沉积技术是纳米颗粒的植入。植入是一种干燥且均匀的沉积技术，最初采用了大量的簇离子源。金簇离子电流不足以植入大面积，因此不适合完整的器官成像。 Nplanter（Ionwerks，Houston，TX）的发展使整个器官组织切片都可以用金属纳米颗粒植入。在这些研究中，银靶被插入粒子源纳米原子50中。这些纳米颗粒（NP）是通过使用磁控溅射从金属表面驱动而产生的，然后通过在改进区中的凝结生长（颗粒的直径为0.5-15 nm）。选择尺寸是用偶联的四极质量滤波器与磁控管耦合的，以便我们可以确保NP大小可重现。目前，我们的直径为6-7 nm，因此我们始终具有植入不同组织切片中植入的相同大小的纳米颗粒，使植入可再现。最后，粒子梁可以在至少400平方米（20 x 20）的最小值上偏离电镜，从而使整个组织截面植入。我们使用的是2-4个单层。 植入每个组织截面需要18分钟。通过使用相同大小的NP颗粒和相同数量的单层。我们已经标准化了植入程序，并使其可重现。因此，可以比较在各个时间植入的组织，因为它们更有可能提供可再现的数据。另外，可以将组织部分重新植入以进行深入分析。到目前为止，我们已经能够连续9次重新植入和重新成像组织部分。每个图像的区域都更深10-15纳米。但是，我们仍在确认深度。单个植入后，我们还能够连续五次成像相同的组织部分。植入器降低了操作员之间的固有可变性，并降低了湿度和温度波动的影响，这使得它在时间，样品和操作员之间保持一致。银植入是一种干方法，因此不会引起溶剂溶解的基质可能发生的任何模糊或分析物迁移。我们发现，用艺术喷枪的手动喷涂是一种相当可重复的方法，用于沉积具有最小脂质破坏的有机矩阵。但是，这可能是一个需要经验丰富的手的大量工作和耗时的过程。在植入时，快速自动化的栅格步骤通过精确覆盖整个植入区域，在基质植入中实现了出色的均匀性。在每个点植入的颗粒量的总控制也有助于提高植入的均匀性和可重复性。可以通过测量来验证白银的数量和覆盖质量。我们还对心脏和肾脏进行了成像，以证明所有组织中不仅是大脑的可重复性。 这种类型的成像对于研究许多器官的疾病动物模型非常有用。 已经对使用AGNP的脂质的脂质类型进行了其他研究。我们的工作表明AGNP是中性脂质（例如神经酰胺（CER），半乳糖酰胺（Galcer），二酰甘油（DAG）和三酰基甘油（TAG）等中性脂质的极好基质。过去，CER和DAG的质谱成像（MSI）使用了M-水质量峰，这是有问题的，因为该质量峰可以由其他复合脂质产生。使用AGNP的启用了仅绘制完整的CER和DAG的MALDI图像，因此没有干扰脂质物种。在负离子模式下，AGNP的产量结果与有机矩阵相似，但具有干涂层方法的额外优势。