Single Extracellular VEsicle Nanoscopy

Abstract

Extracellular vesicles (EVs) and their cargo constitute novel biomarkers. EV subpopulations have been defined not only by abundant tetraspanins (e.g., CD9, CD63 and CD81) but also by specific markers derived from their source cells. However, it remains a challenge to robustly isolate and characterize EV subpopulations. Here, we combined affinity isolation with super‐resolution imaging to comprehensively assess EV subpopulations from human plasma. Our Single Extracellular VEsicle Nanoscopy (SEVEN) assay successfully quantified the number of affinity‐isolated EVs, their size, shape, molecular tetraspanin content, and heterogeneity. The number of detected tetraspanin‐enriched EVs positively correlated with sample dilution in a 64‐fold range (for SEC‐enriched plasma) and a 50‐fold range (for crude plasma). Importantly, SEVEN robustly detected EVs from as little as ∼0.1 μL of crude plasma. We further characterized the size, shape and molecular tetraspanin content (with corresponding heterogeneities) for CD9‐, CD63‐ and CD81‐enriched EV subpopulations. Finally, we assessed EVs from the plasma of four pancreatic ductal adenocarcinoma patients with resectable disease. Compared to healthy plasma, CD9‐enriched EVs from patients were smaller while IGF1R‐enriched EVs from patients were larger, rounder and contained more tetraspanin molecules, suggestive of a unique pancreatic cancer‐enriched EV subpopulation. This study provides the method validation and demonstrates that SEVEN could be advanced into a platform for characterizing both disease‐associated and organ‐associated EV subpopulations.

Size photometry and fluorescence imaging of immobilized immersed extracellular vesicles

“…2). Our setup makes no compromise on fluorescence imaging sensitivity, and we note that our SPFI workflow is compatible with super-resolution imaging 6 , EV subpopulation enrichment via affinity capture surfaces 6 , and cyclic immunofluorescence imaging 14 .…”

“…Multidimensional characterization will be required and could involve multiplex proteomic analysis or 3 incorporation of orthogonal parameters such as EV size. It is furthermore understood that EV diameters in various samples follow inverse power-law distributions where small EVs with diameter 𝑑 < 100 nm are by far the most abundant 6,7 . Consequently, the limit of detection (LOD) of any single EV technology profoundly influences the results.…”

“…The LOD of FCM is commensurate to nanoparticle tracking analysis (NTA) with a label-free LOD of d ≈ 80 nm and which is commonly used to assess EV size distributions. Lowering the detection threshold remains a challenge as nanoparticle scattering scales with d 6 , and lowering the LOD just threefold requires almost a thousand-fold gain in sensitivity. Recently, fluorescence imaging of EVs captured on a surface by widefield or TIRF microscopy 10–12 has gained traction as alternative to FCM due to its accessibility, higher fluorescence sensitivity 13 , and the possibility to manipulate EVs following their immobilization 14 .…”

Advanced Nanoencapsulation-Enabled Ultrasensitive Analysis: Unraveling Tumor Extracellular Vesicle Subpopulations for Differential Diagnosis of Hepatocellular Carcinoma via DNA Cascade Reactions

“…Extracellular vesicles (EVs) encompass a heterogeneous population of phospholipid membrane-enclosed micro- or nanoparticles actively secreted by cells into various body fluids. , Proteomic analyses have revealed that the heterogeneity of EVs arises from the generation of multiple EV subpopulations by both identical and different parental cells in various states. , These subpopulations exhibit diverse surface protein compositions, leading to distinct functional properties . Particularly, tumor-derived EV (tEV) subpopulations serve as critical regulators of intercellular communication, greatly affecting the biology and function of both tumor and microenvironment cells by packaging and transferring bioactive materials , closely associated with primary tumor growth and progression. − Therefore, tEV subpopulations are considered as promising biomarkers for early cancer detection, including HCC. − However, the precise analysis of tEV subpopulations is challenging because of their limited presence amidst normal cell-secreted EVs, free target proteins, and size- and density-overlapping non-EV particles (e.g., protein aggregates, lipoproteins, cell debris) in clinical specimens. − …”

Light-induced Extracellular Vesicle Adsorption

“…These studies often combine complementary characterization tools to classify EVs based on their surface antigens, internal contents, or even physical and mechanical properties[8–10]. Of these techniques, single EV characterization using fluorescence requires efficient EV capture using antibodies, other specific ligands, or nonspecific lipoplexes to effectively concentrate EVs on surfaces[11–13]. Then, total fluorescence or colo-alization of multiple fluorescent probes (e.g.…”