Super-multiplexed fluorescence nanoscopy for imaging-based proteomics

项目总结 原位免疫荧光成像是研究肿瘤的定位、表达水平和 细胞和组织中蛋白质的结构。特别是，多路复用成像揭示了相互作用的网络 这使我们能够了解许多疾病的潜在机制。然而，它已经 由于其极其耗时，执行多路免疫荧光成像一直具有挑战性 工艺简单，成本高，缺乏信号放大功能。共焦可实现的有限空间分辨率 显微镜通常不能揭示复杂的空间组织和确定蛋白质的定位。这里 我们提出了一种超多路免疫荧光纳米显微镜，它能够成像20多个 以纳米级的分辨率在24小时内检测不同的蛋白质。我们将使用DNA条形码的二级纳米体 它们是单价的，开源的，专为定量标记而设计。反复引水洗涤 荧光DNA成像仪将产生高度多路复用的图像。此外，我们还将发展 采用平行化线阵的史无前例的快速受激发射耗尽(STED)显微镜 甜甜圈的横梁。它将具有较大的成像面积和出色的光学切片能力。光子 重新分配、高光谱成像和深度学习将进一步促进基于超分辨率的快速 蛋白质图谱。我们的新生化和光学工具将在不同的生物医学领域发挥关键作用 包括大脑蛋白质组学和癌症图谱。

PROJECT SUMMARY In situ immunofluorescence imaging is a powerful method to study the locations, expression levels and structures of proteins in cells and tissues. In particular, multiplexed imaging reveals the interaction networks of proteins, which allows us to understand the underlying mechanisms of many diseases. However, it has been challenging to perform multiplexed immunofluorescence imaging due to its extremely time-consuming process, high cost and lack of signal amplification. The limited spatial resolution achievable with confocal microscopy often fails to reveal complex spatial organization and to determine localizations of proteins. Here we propose super-multiplexed immunofluorescence nanoscopy that is capable of imaging more than twenty different proteins in 24 hours with nanoscale resolution. We will employ DNA-barcoded secondary nanobodies that are monovalent, open-source and designed for quantitative labeling. Repeated introduction and washing of fluorescent DNA imagers will generate highly multiplexed images. Moreover, we will develop unprecedentedly fast stimulated emission depletion (STED) microscopy that employs a parallelized line array of doughnut beams. It will feature a large imaging area and excellent optical sectioning capability. Photon reassignment, hyperspectral imaging and deep-learning will further facilitate rapid super-resolution-based protein profiling. Our new biochemical and optical tools will play crucial roles in diverse biomedical areas including brain proteomics and cancer profiling.