Spatial proteomics using highly parallel fluorescence hyperspectral and lifetime imaging

使用高度并行荧光高光谱和寿命成像的空间蛋白质组学

• 批准号: 10503477
• 负责人: ENRICO GRATTON
• 金额: $ 55.59万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-22 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10503477
• 关键词: 3-Dimensional; Address; Alzheimer' s Disease; Architecture; Base Sequence; Basic Science; Benchmarking; Biological; Biological Assay; Biological Process; Biology; Biopsy; Cell physiology; Cells; Clinical; Color; Communities; Complementary DNA; Complex; Consumption; Cytometry; DNA Microarray Chip; DNA Probes; Data; Detection; Development; Disease; Equipment; Exhibits; Fluorescence; Homeostasis; Image; Immunofluorescence Immunologic; Immunohistochemistry; Immunooncology; In Situ; Label; Lasers; Mass Spectrum Analysis; Measurement; Mediating; Methods; Modality; Nature; Oligonucleotides; Pathologic; Patient Care; Patients; Performance; Phase; Phenotype; Physiologic pulse; Positioning Attribute; Process; Protein Analysis; Proteins; Proteome; Proteomics; Publishing; Regulation; Reproducibility; Research; Resolution; Sampling; Sensitivity and Specificity; Shapes; Stains; System; Technology; Thick; Thinness; Time; Tissue Model; Tissues; Tumor Tissue; Validation; Visualization; Visualization software; Work; antibody conjugate; base; brain tissue; clinical application; clinically relevant; computerized data processing; cost; cost effective; data visualization; fluorescence imaging; fluorescence lifetime imaging; fluorophore; indexing; instrument; neuroinflammation; new technology; novel; optical spectra; organ growth; patient stratification; personalized diagnostics; personalized medicine; protein biomarkers; protein expression; protein profiling; prototype; scale up; single cell sequencing; spatiotemporal; technological innovation; tissue processing; tool; tumor; two-photon

Abstract Multiplexed spatial profiling of protein markers in cells and tissues is critical to basic research and clinical applications. Unfortunately, we currently lack tools that can rapidly and routinely profile a large number of proteins in situ in large tissues with subcellular resolution in a time and cost-effective fashion. Existing tools for in situ protein analysis including immunohistochemistry and immunofluorescence suffer from low multiplexing because of limited separation of spectral channels. Recent single-cell sequencing methods lack the critical spatial context needed to understand complex heterogeneous samples. Other spatial proteomics methods that are based on serial labeling and imaging, indirect indexed mass spectrometry or sequencing are complicated, time-consuming and expensive. This proposed project will develop a new spatial proteomics technology termed as Phasor S- FLIM that enables direct, simultaneous, high-plex spatial profiling of protein markers in large and thick tissues with just one-round of staining and imaging. Our Phasor S-FLIM system, for the first time, allows true parallel, simultaneous lifetime and spectral detection with phasor analysis to obtain fast, unbiased, high-precision lifetime and spectral data that can be processed in real time. In the proposed work, we will adapt and further develop Phasor S-FLIM for high-plex spatial proteomics applications, including (a) implementation of Pulsed Interleaved Excitation (PIE) dual excitation with 2-photon lasers and sensitive multi-channel GaAsP PMT arrays, enabling exciting and detecting a broad range of fluorophores (Aim 1), (b) development of a novel fluorophore-quencher labeling strategy to generate a large repertoire of probes with orthogonal lifetime and spectrum signatures for high-plex target encoding (Aim 2), and (c) characterization, validation and benchmarking of Phasor S-FLIM for multiplexed spatial protein analysis using broadly relevant biological and clinical tissue models (Aim 3). Once developed, we expect the Phasor S-FLIM can detect at least 30 different protein targets through direct, one round of staining and imaging, in thick (>0.5 mm) tissues, with subcellular resolution (200 nm), and in high imaging throughput (1 x 1 mm2 plane in <15 min), which is currently not possible with existing methods. Upon successful completion of the proposed work, we will have established a working prototype ready to quickly serve the scientific community to address a broad range of biological and clinical questions that are previously impossible or impractical. Our technology can potentially shift current practice in interrogating protein and cellular processes as well as the complexity and systems in biology and disease with high resolution, throughput and scale.

抽象的 细胞和组织中蛋白质标志物的多重空间分析对于基础研究和临床至关重要 申请。不幸的是，我们目前缺少可以快速，常规介绍大量蛋白质的工具 在大型组织中原位，以一段时间和具有成本效益的方式进行亚细胞分辨率。现有的原位工具 蛋白质分析包括免疫组织化学和免疫荧光，因为 光谱通道分离有限。最近的单细胞测序方法缺乏关键的空间环境 需要了解复杂的异质样品。其他基于的空间蛋白质组学方法 串行标记和成像，间接索引质谱或测序是复杂的，耗时的 而且昂贵。这个拟议的项目将开发一种新的空间蛋白质组学技术，称为s-- Flim可以使大小组织中的蛋白质标记物直接，同时，高音空间分析 只需染色和成像。我们的相sor s-flim系统首次允许真正的平行 通过相拟光分析同时使用寿命和光谱检测，以获得快速，无偏的高精度寿命 以及可以实时处理的光谱数据。在拟议的工作中，我们将适应并进一步发展 用于高质量空间蛋白质组学应用的相拟语S-FLIM，包括（a）脉冲交错的实施 激发（PIE）双重激发与2光子激光器和敏感的多通道GAASP PMT阵列 令人兴奋的和检测广泛的荧光团（AIM 1），（b）开发新型荧光团猝灭剂 标记策略以产生具有正交寿命和频谱特征的大量探针曲目 高质量目标编码（AIM 2）和（c）拟法的表征，验证和基准测试 使用广泛相关的生物学和临床组织模型的多重空间蛋白分析（AIM 3）。一次 开发的，我们预计相拟语S-FLIM可以通过直接检测到至少30个不同的蛋白质靶标 在厚度（> 0.5毫米）的组织中，染色和成像圆形分辨率（200 nm），高 成像吞吐量（<15分钟内1 x 1 mm2平面），现有方法目前不可能。之上 成功完成拟议的工作，我们将建立一个工作原型，准备快速服务 科学界解决了以前的广泛生物学和临床问题 不可能或不切实际。我们的技术可以改变当前的审查蛋白质和细胞的实践 具有高分辨率，吞吐量和的生物学和疾病的复杂性和系统 规模。