Project Summary Nanosized extracellular vesicles and particles (EVPs) have been identified as an important means for cells to communicate with neighboring and distant cells. EVPs are actively investigated to understand their roles in cancer, non-invasive disease diagnosis, and therapeutics. One of the most significant urgent challenges to overcome in EVP research is understanding the heterogeneity of EVPs. EVPs are heterogeneous in their size and molecular cargo contents. As a result, single EVP analysis has been identified as crucial to deciphering the heterogeneity of individual EVPs and understanding their biological roles in diverse diseases. As an example, an ongoing scientific question concerns whether the newly discovered extracellular particles called exomeres and supermeres are monolithic nanoparticles enriched with multiple makers such as proteins, RNA and lipids or if they are a distribution of different functionally-active nanoparticles (such as proteins, nucleic acid and lipids) co-isolated together. The widely used analysis techniques such as mass spectrometry are incapable of analyzing individual EVPs and hence these assays mask the impact of the heterogeneity of EVPs, which has made it impossible to address this question and other open questions to date. To select individual EVs for analysis in a non-destructive manner, it is imperative to develop methods for trapping them in solution. Optical tweezers recently recognized with a 2018 Nobel Prize in Physics have been demonstrated as effective approaches for trapping single cells and larger EVs. Unfortunately, the diffraction limit of light precludes their use for the trapping of single nanosized EVs, and the recently discovered exomeres, and supermeres that are only 35 nm and 25 nm in diameter, respectively. This MIRA research program is comprised of a collection of projects designed to develop new optical nanotweezer technologies for high throughput parallelized trapping of single nanosized EVPs combined with enhanced Raman analysis to provide unique information on the global biomolecular composition of individual nanosized EVs, exomeres and supermeres. Subsequently, we will investigate the use of these tools to address ongoing controversies in EV research. First, we will develop a novel optical nanotweezer approach based on nanoplasmonic structures that will enable: (i) parallelized trapping of thousands of single EVPs within seconds; (ii) enhancement and acquisition of Raman signals from single trapped EVPs nondestructively while they are trapped in solution near nanoplasmonic cavities; and (iii) biomolecular component analysis to determine the global biomolecular composition of individual trapped EVPs. Secondly, we will utilize the developed technologies to address ongoing questions in EVP research including whether the newly discovered exomeres and supermeres are monolithic or comprise a diverse distribution of functionally active nanoparticles. The pertinent findings to be ob...