(510e) Characterization of Extracellular Vesicles By Atomic Force Microscopy

Authors: 
Skliar, M. - Presenter, University of Utah
Chernyshev, V., University of Utah
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MS MS 3 10 2019-04-13T00:35:00Z 2019-04-13T00:40:00Z 1 268 1528 12 3 1793 16.00

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Calibri;mso-hansi-font-family:Calibri;mso-bidi-font-family:Calibri">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. In this
presentation we guide 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 (Figure 1) 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 (Figure 2).

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Figure SEQ Figure \* ARABIC 1: AFM images of hydrated MCF-7 exosomes electrostatically immobilized on the modified mica surface. (A) The height image. (B) The corresponding AFM phase image confirms that the grains in the height image are soft nanoparticles, as should be expected for membrane vesicles. (C) The height data for the three vesicles crossed by the line shown in panel (A) illustrate a flattened shape caused by the electrostatic attraction of exosomes to the positively charged surface of the modified mica. (D) The shape distortion is apparent in an enlarged view the immobilized vesicle boxed in panel (A) and its cross section. The phase image of the same vesicle is shown in (E).

 

 


Figure 1: AFM images of hydrated MCF-7 exosomes electrostatically immobilized on the modified mica surface. (A) The height image. (B) The corresponding AFM phase image confirms that the grains in the height image are soft nanoparticles, as should be expected for membrane vesicles. (C) The height data for the three vesicles crossed by the line shown in panel (A) illustrate a flattened shape caused by the electrostatic attraction of exosomes to the positively charged surface of the modified mica. (D) The shape distortion is apparent in an enlarged view the immobilized vesicle boxed in panel (A) and its cross section. The phase image of the same vesicle is shown in (E).
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Figure SEQ Figure \* ARABIC 2: Dimensional characterization of hydrated vesicles immobilized on the surface and the estimation of their globular size in the solution. (A) The distribution of peak heights above the surface (red curve) has the mean equal to 7.9 nm. The area occupied by immobilized exosomes has 69.6 nm average diameter (blue curve). (B) AFM height image for one of the immobilized exosomes illustrates its highly oblate shape caused by electrostatic forces. The globular size of exosomal vesicles in the solution can be estimated by matching volumes enclosed by surface-immobilized and spherical membrane envelopes. (C) The size distribution of globular vesicles in the solution (red curve) was determined from the AFM data of 561 immobilized vesicles. The vesicle sizes in cryo-TEM images (blue curve) are consistent with the AFM results.

 


relative;z-index:251659264"> width:480px;height:491px"> Figure 2: Dimensional characterization of hydrated vesicles immobilized on the surface and the estimation of their globular size in the solution. (A) The distribution of peak heights above the surface (red curve) has the mean equal to 7.9 nm. The area occupied by immobilized exosomes has 69.6 nm average diameter (blue curve). (B) AFM height image for one of the immobilized exosomes illustrates its highly oblate shape caused by electrostatic forces. The globular size of exosomal vesicles in the solution can be estimated by matching volumes enclosed by surface-immobilized and spherical membrane envelopes. (C) The size distribution of globular vesicles in the solution (red curve) was determined from the AFM data of 561 immobilized vesicles. The vesicle sizes in cryo-TEM images (blue curve) are consistent with the AFM results.
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