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

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.