Multifluorescence Single Extracellular Vesicle Analysis by Time-Sequential Illumination and Tracking

Abstract

We demonstrate a fluorescence-based nanoparticle tracking analysis (NTA) system for the characterization of both the size and membrane protein expression of individual extracellular vesicles (EVs). A sheet of lasers with four different wavelengths was sequentially shone onto extracellular vesicles according to a preprogrammed schedule, providing scattering images intercalated by three fluorescent images. The presence of extracellular vesicles was tracked frame by frame from scattering images. Fluorescence-labeled membrane proteins on EVs were detected by comparing scattering and fluorescent images. The tetraspanins (CD9, CD63, and CD81) of individual HEK293 EVs analyzed by both NTA and total internal reflection fluorescence microscopy showed that the proposed NTA system can contribute to the understanding of individual extracellular vesicles.

Ultrasensitive Single Extracellular Vesicle Detection Using High Throughput Droplet Digital Enzyme-Linked Immunosorbent Assay

“…We also compared the performance of DEVA with conventional EV characterization platforms NTA and Nanoview, using aliquots of the same samples processed by our μMD (Figure S9). NTA is a common tool for EV quantification and surface protein analysis . We found, consistent with the literature, the LOD for NTA is ∼ O (10 4 EV/μL) with DR (10 4 –2 × 10 5 EV/μL).…”

“…Accordingly, we developed a platform to measure beads at a high throughput (∼20 million droplets/min) by parallelizing droplet generation, processing, and analysis, achieving a throughput >100× greater than typical in microfluidic systems using only accessible optics (<$1000) and soft-lithography fabrication. We evaluated our technology by first quantifying human neuron derived EVs spiked into PBS and achieved a LOD = 9 EVs/μL, a >100× improvement over standard single EV characterization methods . Moreover, we directly demonstrated the importance of the throughput of our system by showing that the LOD of DEVA improved with an increase in the number of beads up to 10 6 beads.…”

Future of Digital Assays to Resolve Clinical Heterogeneity of Single Extracellular Vesicles

“…Cho et al . designed a multifluorescence single EV analysis by performing nanoparticle tracking analysis (NTA) platform to sequentially track scattering and fluorescence signals frame by frame . The size and expression of membrane proteins (CD9, CD63, and CD81) of single EVs are analyzed by coupling NTA and TIRF.…”

Efficient Labeling of Vesicles with Lipophilic Fluorescent Dyes via the Salt-Change Method

“…Fluorescent labeling of extracellular vesicles (EVs) offers a unique approach to study physical and functional properties of vesicles. For example, tracking diffusion of EVs under fluorescence microscopes can measure the size distribution of EV population, similarly to nanoparticle tracking analysis by light scattering. , More sophisticated techniques also exist for super-resolution imaging, multiplexed measurements, or flow cytometry of EVs. , Lastly, docking of EVs on live cell membranes and their subsequent uptake can be followed in a quantitative manner. − Regardless of the specific properties under investigation, the results can be often confounding due to the underlying heterogeneity in vesicles, in which case the analysis must be conducted at the single-vesicle level to faithfully reconstruct the ensemble properties . Therefore, effective and unbiased, homogeneous labeling with bright fluorescent dyes that visualizes individual vesicles is a prerequisite for EV analysis by fluorescence.…”