Single Extracellular VEsicle Nanoscopy-Universal Protocol (SEVEN-UP): Accessible Imaging Platform for Quantitative...

Extracellular vesicles (EVs), membrane-encapsulated nanoparticles shed from all cells, are tightly involved in critical cellular functions. Moreover, EVs have recently emerged as exciting therapeutic modalities, delivery vectors, and biomarker sources. However, EVs are difficult to characterize, because they are typically small and heterogeneous in size, origin, and molecular content. Recent advances in single EV methods have addressed some of these challenges by providing sensitive tools for assessing individual vesicles; one example is our recently developed Single Extracellular VEsicle Nanoscopy (SEVEN) approach. However, these tools are typically not universally available to the general research community, as they require highly specialized equipment. Here, we show how single EV studies may be democratized via a novel method that employs super-resolution radial fluctuations (SRRF) microscopy and advanced data analysis. SRRF is compatible with a wide range of microscopes and fluorophores. We herein quantified individual EVs by combining affinity isolation (analytical protocol based on SEVEN) with SRRF microscopy and new analysis algorithms supported by machine learning-based EV assessment. Using SEVEN, we first optimized the workflow and validated the data obtained on wide-field and total internal reflection fluorescence microscopes. We further demonstrated that our approach, which we call the SEVEN-Universal Protocol (SEVEN-UP), can robustly assess the number, size, and content of plasma and recombinant EVs. Finally, we used the platform to assess RNA in EVs from conditioned cell culture media. Using SYTO RNASelect dye, we found that 18% of EVs from HEK 293T cells appear to contain RNA; these EVs were significantly larger compared with the general EV population. Altogether, we developed an economical, multiparametric, single EV characterization approach for the research community.