Single Molecule Localization Microscopy for Studying Small Extracellular Vesicles

Abstract

guidelines suggests that EV researchers use the term small EVs (sEVs) for vesicles that are less than 200 nm in diameter. [4] sEVs contain many essential cargo biomolecules such as nucleic acids (DNA, mRNA, microRNA), proteins, and lipids. [1,3,5] sEVs have emerged as a functional mediator for communications between cells in health and disease. [1,6] sEVs derived from tumor cells contain disease-specific proteins, RNA, and double-stranded DNA (dsDNA), thereby representing the disease state and progression. [1,2,[7][8][9] Especially in tumor microenvironment, sEVs transfer their cargo from the tumor to stromal cells. [10,11] sEVs play pathophysiological roles in many other diseases, including neurodegenerative diseases, and various infections. They transmit important biomolecules that regulate many biological processes and influence the immune system. [1,[12][13][14][15] In the last decade, studies dealing with sEV biogenesis pathways and the role of sEVs in health and disease have grown exponentially. However, exploring the released sEVs and interaction of their cargo with cellular biomolecules in the distant recipient cells is significantly hampered by the lack of microscopy studies using improved imaging technology. Most of the current microscopy studies involving sEVs use conventional confocal microscopy to generate either 2D or 3D reconstruction images using Imaris. [11,[16][17][18][19][20][21][22] Although these images provide the information Small extracellular vesicles (sEVs) are 30-200 nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell-to-cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200-300 nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super-resolution microscopy techniques, specifically single-molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.

Standardization Approaches for Extracellular Vesicle Loading with Oligonucleotides and Biologics

“…Advances in super‐resolution microscopy, specifically single‐molecule localization microscopy, with improved fluorophores might also contribute to resolving structural information about LM‐loaded EVs in the future. [ 157 ] In summary, single‐vesicle characterization of loaded EVs is in its infancies and might be limited to differentiate loaded and non‐loaded EVs. The inherent heterogeneity of EVs increases the difficulty to detect differences between subpopulations of loaded EVs.…”

Optical Imaging of Single Extracellular Vesicles: Recent Progress and Prospects

“…Single-molecule and single-particle imaging offer detection sensitivity up to the single probe level, thus can be used to analyze the biological content of individual EVs, making it well-suited for identification of EV subpopulations. As shown in Table , various imaging techniques, including single-molecule/single-particle fluorescence imaging, − single-particle dark-field imaging, − SPR imaging, − and SERS imaging, − have been applied to single EV analysis, and have provided important insights into the structure and composition of EVs as well as the EV-related cellular processes. In this section, we will review the recent developments of the various types of single EV imaging techniques, highlight their advantages and limitations, and summarize the key biological findings obtained using these methods.…”

“…Recently, some researchers have employed highly sensitive microscopic methods, including single-molecule/superresolution fluorescence imaging, − single-particle dark-field imaging, − surface-enhanced Raman scattering (SERS) imaging, − and surface plasmon resonance (SPR) imaging, − to perform single EV analysis. These methods are capable of detecting signals from individual EVs, thus offering unique advantages in studying the heterogeneity of these structures.…”

Nanoarray Enabled Size-Dependent Isolation and Proteomics Profiling of Small Extracellular Vesicle Subpopulations toward Accurate Cancer Diagnosis and Prognosis

“…Small extracellular vesicles (sEVs) are lipid bilayer-bound vesicles with diameters ranging from 30 to 150 nm. , They naturally release from the fusion of multivesicular bodies (MVBs) with the plasma membrane, carrying abundant cargoes such as proteins, nucleic acids, and lipids. − sEVs have become the most extensively investigated vesicles in recent decades due to their crucial roles in regulating various biological processes and mediating intercellular communication. − Growing evidence shows that tumor-derived sEVs carry characteristic biomolecular cargoes in different cancers, and their expression levels are closely related to tumor progression, angiogenesis, and metastasis. − Therefore, sEVs have become the most promising source of biomarkers, which may constitute a “tumor signature” to facilitate the early diagnosis and prognosis of cancers noninvasively. Despite significant efforts in sEV-based liquid biopsy, the phenotypic heterogeneity of sEVs severely impedes their development from proof-of-concept to clinical application.…”

“…S mall extracellular vesicles (sEVs) are lipid bilayer-bound vesicles with diameters ranging from 30 to 150 nm. 1,2 They naturally release from the fusion of multivesicular bodies (MVBs) with the plasma membrane, carrying abundant cargoes such as proteins, nucleic acids, and lipids. 3−5 sEVs have become the most extensively investigated vesicles in recent decades due to their crucial roles in regulating various biological processes and mediating intercellular communication.…”