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

细胞分泌的蛋白质对于了解疾病、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同时观察多个细胞，这使得很难看到细胞之间是如何相互作用的，以及它们之间有什么不同。这些方法不是很敏感，限制了有关蛋白质分泌的信息，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的蛋白质释放。该项目的目的是开发一种名为等离子体增强扩张荧光点(PEEFS)的方法，该方法可以高灵敏度和高精度地对分泌蛋白质进行成像，并准确地测量细胞之间的差异。这项新技术将非常明亮的荧光纳米颗粒与膨胀显微镜相结合，这是一种能够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，该项目还旨在向初中生和高中生介绍生物光子学。其目标是鼓励来自STEM中通常不太有代表性的群体的学生对科学和工程感到自信和兴奋。了解细胞分泌蛋白质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。现有的方法涉及平均数千个细胞，导致与分泌蛋白质的空间分布、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。这些考虑突出了一种简单、超灵敏和高分辨率的方法在单细胞水平上成像和量化蛋白质分泌的迫切需要。该项目的目标是引入和建立等离子体增强的扩展荧光点，用于细胞分泌蛋白质的超灵敏和高分辨率成像和准确定量以及细胞间的异质性。这项新的生物等离子技术依赖于研究人员实验室最近推出的超亮荧光纳米结构等离子体荧光，以及一种非传统的超分辨率技术--膨胀显微镜。具体目标包括：(1)实现五种不同的等离子体荧光并用扩张显微镜测定它们的亮度和兼容性；(2)演示PEEFS并提高等离子体-荧光标记的效率；(3)展示用于探测细胞表面和细胞分泌蛋白质的多色PEEFS。该项目代表着一项革命性的进步，因为它旨在设计和实现一种新的生物等离子技术，以极高的分辨率对细胞分泌的蛋白质进行成像和量化。该项目为等离子、超分辨率显微镜和光学生物成像的研究生、本科生和K-12学生提供独特的培训和教育机会。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.