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.

抽象的 基于质谱的空间分析已实现了广泛的内生的无标签投资 生物分子 - 脂肪，胡椒粉/蛋白质，代谢产物和聚糖 - 完整的生物学和临床标本。 这一领域的快速发展为当前的空间法学研究做出了巨大贡献。然而， 基于MALDI的质谱成像的常规空间分辨率，可以说是最广泛的 当今使用的质谱成像技术在常规质量上大约是数十微米 光谱仪。该分辨率限制不可能在单细胞处对标本的无标签分析的障碍 或现有质谱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已经考虑了成功 生物结构的图像超出了光学系统的内在空间分辨率。这种能力具有 导致生物学和临床实验室中超分辨率荧光显微镜的民主化。 但是，当前的扩展显微镜方案和化学与质谱不兼容 成像或分析。 该提案的主要目的是建立样本扩展的生化过程，以推动 现有的质谱成像管道的空间分辨率为亚细胞状态，即少数 微米及以后。为了实现这一目标，我们将开发样本聚合和消化方案 针对质谱成像进行了优化，其中内源性脂质和蛋白质被束缚至 肿胀聚合物网络，随后可以在MALDI质谱仪上进行分析。我们的 实验方法包括（1）发展组织聚合和 脂质质谱成像的扩展； （2）将管道扩展到多路复用脂质和蛋白质 检测和成像。该提案的成功完成将为社区提供无标签的， 亚细胞分辨率分子成像方式，而无需修改现有仪器。