High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay.
High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay.
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DOI：
10.1016/j.crmeth.2022.100267
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发表时间：
2022-08-22
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分泌的蛋白质参与重要的生理过程。用传统的分析方法，很难绘制单个细胞分泌的蛋白质的空间分布图，研究细胞间分泌的异质性，或者检测低丰度或初期分泌的蛋白质。在这里，我们介绍“荧光点分析”，它使用了一种超亮的纳米粒子等离子体荧光，使蛋白质分泌的高分辨率成像成为可能。我们发现，与传统的荧光标记相比，等离子体荧光的亮度增加了16,000倍，信噪比提高了近30倍。我们展示了不同分泌的细胞因子的高分辨率成像，在单链和光谱多路复用的荧光点试验中，显示了细胞同时分泌多个蛋白质的异质性。使用不同的生化刺激，包括结核分枝杆菌感染，以及各种免疫细胞，如巨噬细胞，树突状细胞(DC)，和DC-T细胞共培养，我们证明了该方法是通用的，简便的，并且广泛适用于增强对单细胞分泌组的时空动态的生物学理解。荧光点技术是研究单细胞蛋白质分泌动力学的一种超灵敏分析方法。荧光点技术为测量细胞间的异质性提供了一种数字化的、可量化的信号。我们证明了它在多种细胞类型和刺激下的适用性。多色血浆荧光调色板允许对分泌的蛋白质进行多路检测。了解单细胞水平上蛋白质分泌的空间和时间动态对于理解众多与健康和疾病相关的生物系统至关重要。然而，目前在单细胞水平上研究蛋白质分泌的方法缺乏空间分辨率。单细胞分泌物的高分辨率成像通常需要昂贵的专门显微镜设备，而这些设备通常是无法获得的。我们开发并验证了一种简单但功能强大的方法，称为“荧光点”分析，以成像单个细胞周围分泌蛋白质的空间分布，以及量化细胞间分泌的异质性的能力。超亮纳米标签能够以数字方式对信号进行量化。在单细胞水平上理解蛋白质分泌的空间和时间动态是至关重要的，但到目前为止仍然具有挑战性。为了应对这一挑战，Seth等人。开发并验证了一种简单但功能强大的名为“荧光点分析”的方法，该方法能够以数字分辨率可视化单细胞蛋白质分泌。
Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the “FluoroDOT assay,” which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome. FluoroDOT is an ultrasensitive assay for studying protein secretion dynamics of single cells FluoroDOT provide a digital, quantifiable signal for measuring cell-to-cell heterogeneity We demonstrate applicability on wide-ranging cell-types and stimuli A multicolor plasmonic-fluor palette allows multiplexed detection of secreted proteins Understanding the spatial and temporal dynamics of protein secretion at a single-cell level is essential to understanding numerous biological systems relevant to health and disease. However, currently available methods for studying protein secretion at the single-cell level lack spatial resolution. High-resolution imaging of single-cell secretion typically requires expensive and specialized microscopy facilities that are not routinely available. We developed and validated a simple yet powerful method called the “FluoroDOT” assay to image the spatial distribution of secreted proteins around single cells, along with the capability to quantify cell-to-cell heterogeneity in secretion. The ultrabright nanolabels enable the quantification of the signals in a digital manner. Understanding the spatial and temporal dynamics of protein secretion at a single-cell level is essential, but up until now remained challenging. To address this challenge, Seth et al. developed and validated a simple yet powerful method called "FluoroDOT assay" that enables the visualization of single-cell protein secretions with digital resolution.