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

Washington University, Saint Louis MO

Investigators

Abstract

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.