Application of Single Extracellular Vesicle Analysis Techniques

Abstract

Extracellular vesicles (EVs) are lipid containers that are actively released by cells and contain complex molecular cargoes. These cargoes include abundant material such as genomes and proteins from cells of origin. They are involved in intercellular communication and various pathological processes, showing excellent potential for diagnosing and treating diseases. Given the significant heterogeneity of EVs in complex physiopathological processes, unveiling their composition is essential to understanding their function. Bulk detection methods have been previously used to analyze EVs, but they often mask their heterogeneity, leading to the loss of valuable information. To overcome this limitation, single extracellular vesicle (SEV) analysis techniques have been developed and advanced. These techniques allow for analyzing EVs’ physical information and biometric molecules at the SEV level. This paper reviews recent advances in SEV detection methods and summarizes some clinical applications for SEV detection strategies.

Insight into the Functional Dynamics and Challenges of Exosomes in Pharmaceutical Innovation and Precision Medicine

“…In response to these limitations, advancements in analytical technologies have facilitated the shift towards single-particle analyses, enabling detailed investigation into the unique roles and properties of individual exosomes [ 149 ]. The guidelines proposed by the Minimal Information for Studies of Extracellular Vesicles (MISEV) advocate for the implementation of at least two single-particle analysis techniques to evaluate both the shape and biological attributes of single exosomes, emphasizing the importance of characterizing these vesicles at the individual level [ 150 ]. The development and refinement of single-exosome analysis techniques aim to address critical challenges associated with exosome research, including their intrinsic heterogeneity, measurement precision, complex biochemical composition, and nanoscale dimensions [ 11 ].…”

“…However, super-resolution fluorescence microscopy demands sophisticated instrumentation and powerful data processing software, often resulting in low throughput [ 158 ]. While fluorescence microscopy techniques are pivotal for elucidating the mechanisms of exosome secretion, characterization, and uptake, the potential of fluorescent markers to interfere with their localization, activity, and functionality cannot be overlooked [ 150 ]. Additionally, lipid markers may exhibit non-specificity in their labeling properties or might aggregate [ 159 ].…”

“…By amalgamating Rayleigh light scattering with sheath flow single-molecule fluorescence detection technology, nFCM markedly enhances the sensitivity of both scattering and fluorescence detection relative to conventional FCM [ 162 ]. Capable of analyzing up to 10,000 particles per minute, this technology permits the multi-parametric quantitative measurement of single vesicles, down to a minimum particle size of 40 nm [ 150 ]. However, the advanced equipment and expertise required for nFCM impose limitations on its widespread clinical application.…”

Exosome-Mediated Antigen Delivery: Unveiling Novel Strategies in Viral Infection Control and Vaccine Design

“…It is worth highlighting that major technological advances now enable specific analysis at the level of single vesicles. These significant developments, regularly reviewed, make it possible to collect extremely valuable precision biometric and molecular information on EVs [ 8 ]. Research in the field has revealed that EVs and their cargoes are highly diverse in nature.…”

Quantitative fluorescent nanoparticle tracking analysis and nano-flow cytometry enable advanced characterization of single extracellular vesicles

“…Over the past decade, we have witnessed a rise, and a great expansion of advanced platforms and tools for analyses of extracellular vesicles (EVs) [1]. From bulk assays, which usually provide high sensitivity but unidimensional measurements, the analyses have reached down to a single particle level, opening the possibility for multiparametric assessment of individual EVs, and deeper understanding of their heterogeneity and complexity [2][3][4][5]. These systems allow simultaneous biophysical and biochemical evaluation of EVs by measuring their size, concentration, charge, morphology, refractive index, and/or composition.…”