Spatial proteomics using highly parallel fluorescence hyperspectral and lifetime imaging

摘要 细胞和组织中蛋白质标志物的多重空间图谱对基础研究和临床至关重要 申请。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。 在大组织中原位，具有亚细胞分辨率，时间和成本效益高。用于现场的现有工具 包括免疫组织化学和免疫荧光在内的蛋白质分析存在着低复制率的问题 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 需要理解复杂的异质样本。其他基于空间蛋白质组学的方法 序列标记和成像、间接指标化质谱学或测序是复杂、耗时的 而且很贵。这个拟议的项目将开发一种新的空间蛋白质组技术，称为相量S- 使大型和厚重组织中的蛋白质标志物能够直接、同时、高复合空间分布的薄膜 只需一轮染色和成像。我们的相量S薄膜系统，第一次允许真正的平行， 使用相量分析同时进行寿命和光谱检测，以获得快速、无偏、高精度的寿命 以及可实时处理的光谱数据。在拟议的工作中，我们将适应和进一步发展 相量S-Flim用于高复杂空间蛋白质组学应用，包括(A)脉冲交错的实现 激发(PIE)双激发，采用双光子激光和灵敏多通道GaAsP PMT阵列，实现 激发和检测广泛的荧光团(目标1)，(B)开发一种新型的荧光团猝灭剂 标记策略，以生成具有正交寿命和光谱签名的大型探针库 高复数目标编码(目标2)，和(C)相量S-Flim的表征、验证和基准 利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 开发出来的，我们预计相量S-薄膜可以通过直接检测至少30个不同的蛋白质靶点，一个 一轮染色和成像，在厚(&gt；0.5 mm)组织中，具有亚细胞分辨率(200 Nm)，在高分辨率下 成像吞吐量(1x1mm2平面在&lt；15分钟内)，这是目前用现有方法无法实现的。vt.在.的基础上 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 科学界解决一系列生物学和临床问题，这些问题以前 不可能的或不切实际的。我们的技术可能会改变目前讯问蛋白质和细胞的做法 过程以及生物学和疾病中的复杂性和系统具有高分辨率、吞吐量和 比例。

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.