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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties. 

Abstract

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging. 

Introduction

Extracellular vesicles (EVs) are present in all body fluids, including blood, urine, saliva, milk, and the amniotic fluid. Exosomes form a district class of EVs differentiated from other EVs by endosomal biogenesis, the markers of the endosomal pathway, and the smallest size among all EVs. The size of exosomes is often reported with substantial variability between studies. The sizing results were found to be method dependent, reflecting the difference in physical principles employed by different analytical techniques to estimate EV sizes1,2. For example, the nanoparticle tracking analysis (NTA) ― the mos....

Protocol

1. Isolation of EVs from a biofluid

  1. Isolate EVs by one of the established methods, such as the differential ultracentrifugation8, precipitation, or size-exclusion chromatography9.
  2. Confirm the presence of expected surface and luminal biomarkers and the absence of biomarkers indicating cross-contamination of the preparation. Confirm the lipid bilayer morphology of the isolated particles by electron microscopy.
    NOTE: When isolatin.......

Representative Results

Surface fixation of EVs is a critical step in the imaging sequence. Electrostatic surface immobilization of exosomes, known to have a negative zeta potential, will robustly occur after the mica’s substrate is modified to have a positive surface charge. Without the treatment with NiCl2 to impart positive surface changes, the immobilization of EVs on the substrate was found to be ineffective. The height image in Figure 10A, acquired in the air after the MCF-7 exosome sample co.......

Discussion

The immobilization of EVs from a biological fluid, surface scanning, and image analysis are the essential steps of the developed protocol for the AFM characterization of EVs in liquid. The number of vesicles amenable to AFM imaging scales with the imaged surface area and the surface concentration of the vesicles immobilized on the substrate. Given a negative zeta potential of EVs and exosomes18, we advocate electrostatic fixation of EVs from liquid samples to the AFM substrate. The immobilization .......

Acknowledgements

The authors acknowledge financial support from the National Science Foundation (award number IGERT-0903715), the University of Utah (Department of Chemical Engineering Seed Grant and the Graduate Research Fellowship Award), and Skolkovo Institute of Science and Technology (Skoltech Fellowship). 

....

Materials

NameCompanyCatalog NumberComments
AFM/STM Controller BrukerMultimode Nanoscope VThis AFM controller supports imaging of biological samples in liquid and air. 
AFM/STM metal specimen discs (10 mm)TedPella16207Metal specimen disc on which a mica disk is attached by a double-sided tape or other means.
AFM/STM Mica discs (10 mm)TedPella50Highest quality grade V1 mica, 0.21mm (0.0085”) thick. Interleaved, in packages of 10. Can be mounted on AFM/STM discs. Available in four diameters
AFM probe for imaging in the airBrukerTESP-V2High quality etched silicon probes for tapping mode and non-contact mode for scanning in the air.
AFM probe for soft sample imaging in liquidBrukerMLCTSoft silicon nitride cantilevers with silicon nitride tips, which are well-suited for liquid operation.  The range in force constants enables users to image extremely soft samples in contact mode as well as high load vs distance spectroscopy.
Double sided tapeSpectrum360-77705Used to fix the mica disk on the metal specimen disc.
ExoQuick-TCSystem BiosciencesEXOTC50A-1ExoQuick-TC is a proprietary polymer-based kit designed for exosome isolation from tissue culture media. 
Glass probe AFM holder for imaging in liquidBruker MTFML-V2This glass probe holder is designed for scanning in fluid with the MultiMode AFM.  The holder can be used in peak force tapping mode, contact mode, tapping mode, and force modulation.  The probe is acoustically driven by a separate piezo oscillator for larger amplitude modulation.  The holder is supplied with two ports, required fittings, and accessories kit for adding and removing fluids.
GwyddionCzech Metrology Institute.Version 2.52Open Source software for visualization and analysis of data fields obtained by scanning probe microscopy techniques.
Lint-free blotting paperGE Healthcare Whatman Grade GB003 Blotting PaperUse this blotting paper to remove NiCl2 after the modification of the mica's substrate.  
Lint-free cleanroom wipesTexwipeAlphaWipe TX1004Use these polyester wipes for surface cleaning. 
Nickel(II) chloride (NiCl2)Sigma-Aldrich339350Powder used to make 10 mM NiCl2 in DI water
Phosphate Buffered Saline (1x)Gibco10010023PBS, pH 7.4

References

  1. Chernyshev, V. S., et al. Size and shape characterization of hydrated and desiccated exosomes. Analytical and Bioanalytical Chemistry. 407, 3285-3301 (2015).
  2. Ramirez, M. I., et al. Technical challenges of working with extrace....

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Extracellular VesiclesAtomic Force MicroscopyAFM ImagingElectrostatic ImmobilizationMica SurfaceHydrated SamplesDesiccated SamplesCryoTEMNanoparticle Tracking Analysis

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