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

基本信息

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
项目类别：
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&apos 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

项目摘要

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记物的多重空间分析对于基础研究和临床至关重要应用程序。不幸的是，我们目前缺乏能够快速、常规地分析大量蛋白质的工具以时间和成本效益的方式在大组织中进行亚细胞分辨率的原位分析。现有的现场工具蛋白质分析（包括免疫组织化学和免疫荧光）的多重性较低，因为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本。其他基于空间蛋白质组学的方法连续标记和成像、间接索引质谱或测序复杂且耗时而且很贵。该拟议项目将开发一种新的空间蛋白质组学技术，称为 Phasor S- FLIM 可对大而厚的组织中的蛋白质标记物进行直接、同步、高复杂度的空间分析只需一轮染色和成像。我们的 Phasor S-FLIM 系统首次实现了真正的并行、通过相量分析同时进行寿命和光谱检测，以获得快速、无偏差、高精度的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 Phasor S-FLIM 适用于高复杂空间蛋白质组学应用，包括 (a) 脉冲交错的实施使用 2 光子激光器和灵敏的多通道 GaAsP PMT 阵列进行激发 (PIE) 双激发，使激发并检测多种荧光团（目标 1），(b) 开发新型荧光团猝灭剂标记策略，生成大量具有正交寿命和光谱特征的探针高复数目标编码（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和临床组织模型进行多重空间蛋白质分析（目标 3）。一次开发完成后，我们预计 Phasor S-FLIM 可以通过直接检测至少 30 种不同的蛋白质靶标，一种在厚（>0.5 mm）组织中进行一轮染色和成像，具有亚细胞分辨率（200 nm），并且在高成像吞吐量（1 x 1 mm2 平面<15 分钟），这是目前现有方法无法实现的。之上成功完成建议的工作后，我们将建立一个工作原型，准备快速服务科学界解决以前广泛的生物学和临床问题不可能或不切实际的。我们的技术有可能改变当前研究蛋白质和细胞的实践具有高分辨率、通量和精度的生物学和疾病过程以及复杂性和系统规模。