Review
Microscopy approaches to study extracellular vesicles

https://doi.org/10.1016/j.bbagen.2020.129752Get rights and content

Highlights

  • Microscopy techniques span from single molecule localization to live cell imaging.

  • Here we provide information to plan experiments using these imaging techniques.

  • Imaging is helping to decipher many aspects of EV biology.

  • Many consideration valid to study EVs can be generalized to other nanovectors.

Abstract

Background

Extracellular vesicles (EVs) have drawn the attention of both biological researchers and clinical physicians due to their function in mediating cell-to-cell communication and relevance as potential diagnostic markers. Since their discovery, the small size and heterogeneity of EVs has posed a hindrance to their characterization as well as to the definition of their biological significance.

Scope of the review

Recent technological advances have considerably expanded the tools available for EV studies. In particular, the combination of novel microscope setups with high resolution imaging and the flexibility in EV labelling allows for the precise detection and characterization of the molecular composition of single EVs. Here we will review the microscopy techniques that have been applied to unravel the mechanism of EV-mediated intercellular communication and to study their molecular composition.

Major conclusions

Microscopy technologies have largely contributed to our understanding of molecular processes, including EV biology. As we discuss in this review, careful experimental planning is necessary to identify the most appropriate technique to use to answer a specific question.

General significance

The considerations regarding microscopy and experimental planning that are discussed here are applicable to the characterization of other small structures, including synthetic nanovectors and viruses.

Introduction

Extracellular vesicles (EVs) are 50–500 nm membrane-bound structures released by both prokaryotic and eukaryotic cells [1]. EVs retain ligands on their surface and proteins, nucleic acids, and metabolites in their lumen that reflect the composition of their cell of origin. Currently, two main types of EVs are described: ectosomes (also known as “shedding vesicles”), that are released directly from the cell surface, and exosomes, that are secreted upon fusion of multivesicular bodies (MVBs) with the plasma membrane [2]. Although EVs were first visualized by electron microscopy in the 1960s [3,4], it is only within the last decade that researchers have begun to deeply investigate EV biology.

The renewed interest in EVs is due to two reasons: first, EVs have great potential as biomarkers since they reach body fluids after their release into the extracellular space. The development of techniques to efficiently probe EVs originating from pathological cells can contribute to the establishment of novel tests to monitor disease progression [5]. Second, EVs mediate cell-to-cell communication by stimulating receptors on the surface of acceptor cells and/or by releasing their content into the cytosol of the recipient cells [6]. Understanding the molecular machineries involved in these processes can aid in the development of a new generation of low-immunogenic drug delivery tools [7,8].

Over the years, many approaches have been developed to study EV-mediated intercellular communication. Often the information gained from these experiments is based on phenotypic modifications of the acceptor cells upon EV administration, including changes in morphology, proliferation, and migration competence. These observations are generally accompanied by proteomic and transcriptomic analyses to accomplish two goals: to confirm that EVs are enriched in molecules that can confer a specific phenotype to the acceptor cells, and to characterize the expected molecular modification in the recipient cells upon exposure to EVs. Although valid, these approaches are limited to in-bulk analyses of purified EVs and an average estimate of the modifications induced in the acceptor cells.

Recent developments in other areas of cell biology have shown that in-bulk approaches can be complemented effectively by microscopy experiments. Contemporary advancement of imaging technologies (such as the advent of highly sensitive detectors and the creation of high power lasers for fluorescent excitation) resulted in super-resolution techniques with the capacity to both identify protein localization with extreme precision and estimate the number of molecules retained in a single fluorescent object. The increasing availability of these high-powered imaging tools within a modest budget range has substantially expanded the ability to characterize the molecular composition of EVs while assessing their size with high precision [9]. For example, cancer-specific markers have been detected on single EVs isolated from body fluids [10] that often lack the concentration of EVs required for analyzing their contents by conventional techniques (OMICS, immunoblotting, PCR, flow cytometry, etc.). For this reason, the application of these powerful technologies to the diagnostic sector is promising. We envision that imaging-based platforms will play a significant role in understanding the biological principles of EV-mediated cell-to-cell communication and in increasing the efficiency of EV-based diagnostics.

In this review article, we will explore classic approaches of fluorescence microscopy as well as new, cutting-edge imaging techniques that have been successfully applied to the study of EVs. Moreover, throughout the paper we will discuss potential caveats and challenges in the design of microscopy-based EV studies.

Section snippets

EV labelling approaches

In order to be detected by fluorescence microscopy, EVs need to be stained. EV labelling is achieved by two approaches that hereafter we will refer to as protein- or lipid-based, depending on whether the target molecules of the labelling are proteins or lipids. The fluorescent markers used for labelling EVs have a variety of origins since they can be proteins, small organic molecules, or quantum dots. In addition, these labels can be conjugated to nanobodies or antibodies that can modify the

Fixed-cell microscopy

The majority of studies focused on understanding EV-cell interactions have been performed by fixing the cells after treating with EVs labelled by protein- or lipid-based methods. The main drawback of imaging fixed samples is that the observation is limited to a specific time frame, consequently giving no information about the dynamics of the process. Additionally, fixation with paraformaldehyde and antibody staining may introduce artifacts. However, the advantage of fixed samples is that they

Imaging techniques to study EV biogenesis and release

In addition to characterizing the mechanisms of EV internalization, it is also relevant to understand the contributing factors to EV biogenesis and release, which is an aspect of EV biology still poorly explored.

The formation of shedding vesicles/ectosomes from the plasma membrane has been investigated by imaging fluorescent cells in real time. Since EV release is a constitutive process that occurs slowly, cells are generally treated with various agents, such as ATP, to favor the production of

Light Sheet Fluorescence Microscopy (LSFM): a 3D approach for EV studies in living cells

One of the most innovative imaging technologies that has been developed in the last decade is Light Sheet Fluorescence Microscopy (LSFM). LSFM refers to an array of setups that originated from Orthogonal Plane Fluorescence Optical Sectioning (OPFOS) [59] and Selective Plane Illumination Microscopy (SPIM) [60]. In these microscopes the illumination and detection paths are uncoupled, making it possible to optically section a specimen by using a sheet of light that excites only a thin slice of the

Imaging approaches for the analysis of purified EVs

EVs are heterogeneous populations that differ in origin and composition. By applying specific microscopy approaches, it is possible to define the molecular profile of each single EV. This information is important to define EV origin as well as to foster the development of sensitive diagnostic tools that can detect disease specific markers on EVs collected from body fluids of patients (liquid biopsy). Here we will overview successful applications of these approaches.

Concluding remarks

The ability to precisely characterize the surface markers, internal cargoes, and size of single EVs as well as study their fate after cellular uptake are all equally important aspects for gaining a holistic understanding of EV involvement in physiological and pathological conditions. Recent innovations in sample staining and advances in both conventional and super-resolution microscopy have opened up new opportunities to explore various cellular processes with high detail, including EVs. We

Funding

Emanuele Cocucci was supported by funding provided by Ohio Cancer Research via the McCurdy/Kimball Midwest Research Fund. Federico Colombo was supported by an American-Italian Cancer Foundation Post-Doctoral Research Fellowship.

Declaration of competing interests

None.

References (66)

  • P. Wolf

    The nature and significance of platelet products in human plasma

    Br. J. Haematol.

    (1967)
  • O. Behnke

    Electron microscopical observations on the surface coating of human blood platelets

    J. Ultrasruct. Res.

    (1968)
  • E. Colombo et al.

    Microvesicles: novel biomarkers for neurological disorders

    Front. Physiol.

    (2012)
  • L. Gangoda et al.

    Extracellular vesicles including exosomes are mediators of signal transduction: are they protective or pathogenic?

    Proteomics

    (2015)
  • B. György et al.

    Therapeutic applications of extracellular vesicles: clinical promise and open questions

    Annu. Rev. Pharmacol. Toxicol.

    (2015)
  • S. Raimondo et al.

    Extracellular vesicles as biological shuttles for targeted therapies

    Int. J. Mol. Sci.

    (2019)
  • C. Chen et al.

    Visualization and intracellular dynamic tracking of exosomes and exosomal miRNAs using single molecule localization microscopy

    Nanoscale

    (2018)
  • K.M. Lennon et al.

    Single molecule characterization of individual extracellular vesicles from pancreatic cancer

    J. Extracell. Vesicles

    (2019)
  • D.K. Jeppesen et al.

    Reassessment of exosome composition

    Cell

    (2019)
  • M. Gabrielli et al.

    Active endocannabinoids are secreted on extracellular membrane vesicles

    EMBO Rep.

    (2015)
  • C.P. Lai et al.

    Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters

    Nat. Commun.

    (2015)
  • F. Colombo et al.

    Cytokines stimulate the release of microvesicles from myeloid cells independently from the P2X7 receptor/acid sphingomyelinase pathway

    Front. Immunol.

    (2018)
  • G. Corso et al.

    Systematic characterization of extracellular vesicles sorting domains and quantification at the single molecule–single vesicle level by fluorescence correlation spectroscopy and single particle imaging

    J. Extracell. Vesicles

    (2019)
  • C.M. Termini et al.

    Tetraspanins function as regulators of cellular signaling

    Front. Cell Dev. Biol.

    (2017)
  • F. Bianco et al.

    Acid sphingomyelinase activity triggers microparticle release from glial cells

    EMBO J.

    (2009)
  • A. Mondal et al.

    Effective visualization and easy tracking of extracellular vesicles in glioma cells

    Biol. Proced. Online

    (2019)
  • K. Lee et al.

    Multiplexed profiling of single extracellular vesicles

    ACS Nano

    (2018)
  • C. Chen et al.

    Imaging and intracellular tracking of cancer-derived exosomes using single-molecule localization-based super-resolution microscope

    ACS Appl. Mater. Interfaces

    (2016)
  • C. Verderio et al.

    Myeloid microvesicles are a marker and therapeutic target for neuroinflammation

    Ann. Neurol.

    (2012)
  • L. Garzetti et al.

    Activated macrophages release microvesicles containing polarized M1 or M2 mRNAs

    J. Leukoc. Biol.

    (2014)
  • N. Nishida-Aoki et al.

    Disruption of circulating extracellular vesicles as a novel therapeutic strategy against cancer metastasis

    Mol. Ther.

    (2017)
  • S. Zhang et al.

    Size-dependent endocytosis of nanoparticles

    Adv. Mater.

    (2009)
  • S. Zong et al.

    Single molecule localization imaging of exosomes using blinking silicon quantum dots

    Nanotechnology

    (2018)
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