Plasmon-enhanced Expansion FluoroSpot for Imaging and Quantifying Single Cell Protein Secretion

细胞分泌蛋白对于了解疾病、开发新的诊断技术和未来的个性化药物非常重要。目前监测分泌蛋白的方法是将许多细胞放在一起观察，这使得很难看到细胞如何相互作用以及它们之间的差异。这些方法不是很敏感，限制了关于蛋白质分泌的信息，特别是在细胞受到刺激后。这些挑战表明，迫切需要一种简单、高灵敏度和高分辨率的方法来观察单个细胞水平上的蛋白质释放。该项目的目的是开发一种称为等离子体增强膨胀荧光斑（PEEFS）的方法，该方法可以对分泌蛋白进行高灵敏度和高精度成像，并准确测量细胞之间的差异。这项新技术将非常明亮的荧光纳米粒子与扩展显微镜相结合，这是一种实现高分辨率光学成像的技术。除了培养研究生和本科生外，该项目还旨在向初高中学生介绍生物光子学。其目标是鼓励来自通常在STEM中没有充分代表的群体的学生对科学和工程感到自信和兴奋。了解细胞分泌蛋白的时空动态在许多生命科学学科中是必不可少的，包括免疫学、肿瘤学和干细胞生物学。现有的方法涉及对成千上万的细胞进行平均，导致与分泌蛋白的空间分布、细胞间异质性和细胞间相互作用相关的信息丢失。此外，由于现有技术的低灵敏度，关于蛋白质分泌动力学的信息很少，特别是在细胞刺激后的早期时间点和低水平刺激下。这些考虑突出了对一种简单、超灵敏和高分辨率方法的迫切需求，以在单细胞水平上成像和量化蛋白质分泌。该项目的目标是引入和建立等离子体增强膨胀荧光点，用于超灵敏和高分辨率成像，准确定量细胞分泌蛋白和细胞间异质性。这种新颖的生物等离子体技术依赖于一种超明亮的荧光纳米结构，等离子体荧光，最近由研究人员的实验室推出，以及扩展显微镜，一种非常规的超分辨率技术。具体目标包括：(1)利用扩增显微镜实现五种不同的等离子体荧光并确定其亮度和相容性；(2)演示PEEFS并提高等离子体荧光标记效率；(3)演示用于探测细胞表面和细胞分泌蛋白的多色PEEFS。该项目代表了一个变革性的进步，它旨在设计和实现一种新的生物等离子体技术，以极高的分辨率对细胞分泌的蛋白质进行成像和量化。该项目为研究生、本科生和K-12学生提供了等离子体、超分辨率显微镜和光学生物成像交叉领域的独特培训和教育机会。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Cell secreted proteins are important for understanding disease and for developing new diagnostic technologies and future personalized medicines. Current methods for monitoring secreted proteins observe many cells together, which makes it difficult to see how cells interact with each other and how they differ. These methods are not very sensitive, limiting information about protein secretion, especially after cells are stimulated. These challenges point to an urgent need for a simple, highly sensitive, and high-resolution method to observe protein release at the level of individual cells. The aim of this project is to develop a method called Plasmon-Enhanced Expansion FluoroSpot (PEEFS), which can image secreted proteins with high sensitivity and precision, and accurately measure differences between cells. This new technology combines a very bright fluorescent nanoparticle with expansion microscopy, a technique that enables high resolution optical imaging. In addition to training graduate and undergraduate students, the project aims to introduce middle and high school students to biophotonics. The goal is to encourage students from groups not usually well-represented in STEM to feel confident and excited about science and engineering.Understanding the spatial and temporal dynamics of cell-secreted proteins is essential in numerous life science disciplines, including immunology, oncology, and stem cell biology. Existing methods involve averaging many thousands of cells, resulting in the loss of information related to the spatial distribution of secreted proteins, cell-to-cell heterogeneity, and cell-cell interaction. Furthermore, due to the low sensitivity of the existing techniques, there is little information on the kinetics of protein secretion, particularly at early time points after cell stimulation and under low levels of stimulation. These considerations highlight the critical need for a simple, ultrasensitive, and high-resolution method to image and quantify protein secretion at a single-cell level. The goal of the project is to introduce and establish Plasmon-Enhanced Expansion FluoroSpot for ultrasensitive and high-resolution imaging and accurate quantification of cell-secreted proteins, and cell-to-cell heterogeneity. This novel bioplasmonic technology relies on an ultrabright fluorescent nanoconstruct, plasmonic-fluor, recently introduced by the investigator’s lab, and expansion microscopy, an unconventional super-resolution technique. Specific objectives include: (1) realizing five distinct plasmonic-fluors and determining their brightness and compatibility with expansion microscopy, (2) demonstrating PEEFS and improving plasmonic-fluor labeling efficiency, and (3) demonstrating multi-color PEEFS for probing cell surface and cell-secreted proteins. The project represents a transformative advance in that it aims to design and realize a novel bioplasmonic technology to image and quantify cell-secreted proteins at extremely high resolution. The project offers unique training and educational opportunities to graduate, undergraduate, and K-12 students at the intersection of plasmonics, super-resolution microscopy, and optical bioimaging.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.