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

用于单细胞蛋白质分泌成像和定量的等离激元增强扩增 FluoroSpot

• 批准号: 2316285
• 负责人: Srikanth Singamaneni
• 金额: $ 45万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2316285&HistoricalAwards=false
• 关键词: Plasmon; enhanced; Expansion; FluoroSpot; Imaging

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.

细胞分泌的蛋白质对于理解疾病和开发新的诊断技术和未来的个性化药物非常重要。目前用于监测分泌蛋白的方法观察到许多细胞在一起，这使得很难看到细胞如何相互作用以及它们如何不同。这些方法不是很灵敏，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扩增荧光点（PEEFS）的方法，该方法可以以高灵敏度和精度对分泌蛋白进行成像，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颗粒与扩展显微镜相结合，这是一种能够实现高分辨率光学成像的技术。除了培训研究生和本科生，该项目旨在向初中和高中学生介绍生物光子学。我们的目标是鼓励那些在STEM中通常没有很好代表性的学生对科学和工程充满信心和兴奋。了解细胞分泌蛋白质的空间和时间动力学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。现有的方法涉及对数千个细胞进行平均，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、超灵敏和高分辨率的方法的迫切需求，该方法可以在单细胞水平上对蛋白质分泌进行成像和定量。该项目的目标是引入和建立Plasmon增强的Expansion FluoroSpot，用于超灵敏和高分辨率成像以及细胞分泌蛋白质和细胞间异质性的准确定量。这种新的生物等离子体技术依赖于一种超亮荧光纳米结构，等离子体荧光，最近由研究人员的实验室，和扩展显微镜，一种非传统的超分辨率技术。具体目标包括：（1）实现五种不同的等离子体荧光并确定它们的亮度和与扩增显微镜的兼容性，（2）展示PEEFS并提高等离子体荧光标记效率，以及（3）展示多色PEEFS用于探测细胞表面和细胞分泌的蛋白质。该项目代表了一个变革性的进步，因为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定量。该项目为研究生、本科生和K-12学生在等离子体、超分辨率显微镜和光学生物成像领域提供了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价值和更广泛的影响审查标准进行评估，被认为值得支持。