A biochemical approach towards subcellular, label-free molecular imaging

亚细胞、无标记分子成像的生化方法

• 批准号: 10686627
• 负责人: Ruixuan Gao
• 金额: $ 137.3万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686627
• 关键词: Area; Biochemical; Biochemical Process; Biological; Biological Markers; Cells; Chemistry; Clinical; Communities; Democracy; Detection; Development; Digestion; Fluorescence Microscopy; Goals; Image; Imaging Techniques; Investigation; Label; Laboratories; Lipids; Maps; Mass Spectrum Analysis; Microscopy; Optics; Pathology; Peptides; Polymers; Polysaccharides; Proteins; Protocols documentation; Public Health; Research; Resolution; Sampling; Specimen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Subcellular Anatomy; Swelling; System; Tissues; disease diagnosis; fluorescence imaging; imaging modality; instrumentation; mass spectrometer; mass spectrometric imaging; meter; molecular imaging; novel strategies; polymerization; success; ultra high resolution

ABSTRACT Mass spectrometry-based spatial analysis has enabled label-free investigation of a broad range of endogenous biomolecules—lipids, peptides/proteins, metabolites, and glycans—in intact biological and clinical specimens. Rapid development in this area has contributed enormously to the current spatial omics research. However, the routine spatial resolution of MALDI-based mass spectrometry imaging, arguably one of the most widely used mass spectrometry imaging techniques today, is about tens of micrometers on a conventional mass spectrometer. This resolution limit imposes a roadblock towards label-free analyses of specimens at single-cell or subcellular level in existing mass spectrometry laboratories. Recent development of expansion microscopy has seen considerable success in generating fluorescence images of biological structures beyond the intrinsic spatial resolution of an optical system. Such capability has led to democratization of super-resolution fluorescence microscopy in biological and clinical laboratories. However, current expansion microscopy protocols and chemistries are not compatible with mass spectrometry imaging or analyses. The main objective of this proposal is to establish a biochemical process of sample expansion to push the spatial resolution of existing mass spectrometry imaging pipelines to the subcellular regime, namely, to a few micrometers and beyond. Towards this goal, we will develop a sample polymerization and digestion protocol optimized for mass spectrometry imaging, in which the endogenous lipids and proteins are tethered to a swelling polymer network and can be subsequently analyzed on a MALDI mass spectrometer. Our experimental approaches include (1) development of a biochemical pipeline of tissue polymerization and expansion for lipid mass spectrometry imaging; (2) extension of the pipeline to multiplexed lipid and protein detection and imaging. Successful completion of this proposal will provide the community with a label-free, subcellular-resolution molecular imaging modality without modifying the existing instrumentation.

摘要 基于质谱学的空间分析使得能够对广泛的内源 完整的生物和临床标本中的生物分子--脂类、多肽/蛋白质、代谢物和多糖。 这一领域的快速发展为当前的空间组学研究做出了巨大贡献。然而， 基于MALDI的质谱学成像的常规空间分辨率，可以说是最广泛的 今天使用的质谱学成像技术，大约是传统质量的几十微米 分光计。这一分辨率限制给单细胞标本的无标记分析设置了障碍 或在现有的质谱学实验室中的亚细胞水平。 膨胀显微镜的最新发展在产生荧光方面取得了相当大的成功 超出光学系统固有空间分辨率的生物结构的图像。这种能力具有 导致了生物和临床实验室中超分辨率荧光显微镜的民主化。 然而，当前的膨胀显微镜方案和化学方法与质谱学不兼容。 成像或分析。 这项建议的主要目标是建立一个样品扩大的生化过程，以推动 现有的质谱学成像管道的空间分辨率达到亚细胞状态，即几个 微米甚至更远。为了实现这一目标，我们将开发一种样品聚合和消化方案 针对质谱学成像进行了优化，在质谱学成像中，内源性脂质和蛋白质被拴在一个 溶胀聚合物网络，随后可在MALDI质谱仪上进行分析。我们的 实验方法包括：(1)组织聚合生化管道的研制 用于脂质谱成像的扩展；(2)将管道扩展到多种脂类和蛋白质 检测和成像。这项提案的成功完成将为社区提供一种无标签、 在不修改现有仪器的情况下，采用亚细胞分辨率的分子成像方式。