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: 10707993
• 负责人: ENRICO GRATTON
• 金额: $ 32.39万
• 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/10707993
• 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; Dimensions; Disease; Equipment; Exhibits; Fluorescence; Homeostasis; Image; Immunofluorescence Immunologic; Immunohistochemistry; Immunooncology; In Situ; Label; Lasers; Maps; Mass Spectrum Analysis; Measurement; Mediating; Methods; Modality; Nature; Oligonucleotides; Pathologic; Patient Care; Patients; Performance; Phenotype; Physiologic pulse; Positioning Attribute; Process; Protein Analysis; Proteins; Proteome; Proteomics; Publishing; Regulation; Reproducibility; Research; Resolution; Sampling; Shapes; Specific qualifier value; Specificity; Stains; System; Technology; Thick; Thinness; Time; Tissue Model; Tissues; Tumor Tissue; Validation; Visualization; Visualization software; Work; antibody conjugate; 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.

摘要 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应用至关重要。 应用.不幸的是，我们目前缺乏能够快速和常规地分析大量蛋白质的工具， 以时间和成本有效的方式在大组织中原位进行亚细胞解析。现场现有工具 包括免疫组织化学和免疫荧光的蛋白质分析受到低复用的影响， 光谱通道的有限分离。最近的单细胞测序方法缺乏关键的空间背景 需要了解复杂的异质样品。其他空间蛋白质组学方法， 连续标记和成像、间接索引质谱或测序是复杂、耗时的 而且很贵。该项目将开发一种新的空间蛋白质组学技术，称为Phasor S- FLIM可在大而厚的组织中直接、同时、高倍数地进行蛋白质标记物的空间分析 只需一轮染色和成像。我们的Phasor S-FLIM系统首次允许真正的并行， 使用相量分析同时进行寿命和频谱检测，以获得快速、无偏、高精度的寿命 以及可以真实的处理的光谱数据。在提出的工作中，我们将适应并进一步发展 用于高复杂度空间蛋白质组学应用的相位器S-FLIM，包括（a）脉冲交错 激发（PIE）双激发，具有2光子激光器和灵敏的多通道GaAsP PMT阵列， 激发和检测宽范围的荧光团（目标1），（B）开发新的荧光团猝灭剂 标记策略，以生成具有正交寿命和光谱特征的大量探针， 高复杂目标编码（目标2），以及（c）Phasor S-FLIM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进行多重空间蛋白质分析（Aim 3）。一旦 我们预计Phasor S-FLIM可以通过直接检测至少30种不同的蛋白质靶点， 圆染色和成像，在厚（>0.5 mm）组织中，具有亚细胞分辨率（200 nm），在高分辨率组织中， 成像吞吐量（在<15分钟内1 × 1 mm 2平面），这在现有方法中是不可能的。后 成功地完成了拟议的工作，我们将建立一个工作原型，准备迅速服务 科学界解决广泛的生物和临床问题，以前是 不可能或不切实际。我们的技术可以潜在地改变目前在询问蛋白质和细胞的实践， 过程以及生物学和疾病中的复杂性和系统，具有高分辨率，高通量和高效率。 规模