Imaging of Extracellular Vesicles by Atomic Force Microscopy

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. 

This protocol outlines simple preparation for AFM imaging of extracellular vesicles in hydrated and desiccated forms, their electrostatic immobilization, surface scanning, vesical identification, and data analysis and interpretation. The main advantage of this technique is the convenient electrostatic fixation of vesicles on the skin's surface and the post imaging analysis to account for shape distortion caused by immobilization. The obtained vesicles sizing results are consistent with the Gold Standard CryoTEM Imaging which remains costly and challenging technique.

If you're a new AFM user, begin with the characterization of dry samples before proceeding to hydrated samples. Because there are numerous additional factors that can impact hydrated sample acquisition. To begin, isolate extracellular vesicles from a bio-fluid as described in the accompanying text protocol.

Next, firmly attach a mica disc to a magnetic stainless steel specimen disc. Cleave the mica disc by using a sharp razor to expose a new layer of material. At room temperature, treat the top surface of mica for ten seconds with one hundred micro-liters of a ten millimolar nickel II chloride solution.

This modifies the surface's charge from negative to positive. Blot the nickel II chloride solution with a lint free wipe or blotting paper. Then, wash the mica surface three times with deionized water and dry it with a stream of dry nitrogen.

Place the AFM specimen disc with the attached surface modified mica into a petri dish. Next, dilute the exosomes with PBS to obtain a concentration between four and forty-billion particles per milliliter of solution. Validate the diluted particle concentration using nano particle tracking analysis.

Form a sessile drop on the surface of the mica by emptying one-hundred microliters of the diluted exosome solution from a pipette. Then place the lid on the petri dish and seal it with parafen film to reduce sample evaporation. Incubate in the refrigerator for twelve hours.

After incubation, aspirate 80 to 90 percent of the sample carefully without disturbing the surface. At this point, the exosomes will be electrostatically immobilized on the mica substrate. When imaging hydrated samples, rinse the surface three times with PBS.

Take care to keep the sample hydrated throughout the rinsing process. After washing the mica surface with PBS, remove 80 to 90 percent of the liquid and pipette forty microliters of fresh PBS to cover the sample. The hydrated sample is ready for imaging.

When imaging the desiccated EV's, remove the salts from the surface by rinsing the substrate three times with deionized water. After aspirating as much liquid as possible, without touching the surface, dry the rest with a stream of dry nitrogen. To image the desiccated extracellular vesicles, select a cantilever designed for scanning in the air in tapping and non-contact imaging modes and mount it onto the probe holder.

Place the sample onto the AFM stage. The magnetic stainless steel specimen disc will immobilize the sample on the stage. Place the probe holder into the AFM.

Allow time for the preparation and the stage to equilibrate thermally. Use the tapping mode to scan an area that is 5x5 microns rastered in 512 lines at a scan rate of one hertz. Acquire both the height and phase images as they provide complimentary information on the topography and the surface properties of the sample.

Scan time will increase with an imaged area and the number of lines selected to form the image, but decrease with the scan rate. Since fast scan rates may impact the image quality, the speed of rastering should be a balance between acquisition time and image quality. To image hydrated vesicles, select a cantilever that is appropriate for scanning soft, hydrated samples and mount the cantilever onto a probe holder designed for scanning in liquids.

Wet the tip of the cantilever with PBS to reduce the likelihood of introducing air bubbles to the liquid during scanning. Then, immobilize the sample onto the AFM stage. Once the sample thermally equilibrates, image the hydrated mica surface in tapping mode.

Acquire both the height and phase images. To analyze the images taken, first go to Data Process'select SPM modes, followed by Tip'and choose Model Tip'Select the geometry and the dimensions of the tip used to scan the sample and click OK'Correct the tip erosion artifacts by performing the surface reconstruction. Open the image.

From the menu, select Data Process'then select SPM Modes'followed by Tip'then choose Surface Reconstruction'and click OK'Next, select Data Process, followed by Level'and choose Plane Level'to align the imaging plane and to match the laboratory XY plane by removing the tilt in the substrate from the scan data. Align rows in the image by selecting Data Process'followed by Correct Data'and then choose Align Rows'Several alignment options are available including median which is an algorithm that finds an average height of each scan line and subtracts it from the data. Next, go to Data Process'followed by Correct Data'and choose Remove Scars'This will remove common scanning errors known as Scars'To align the mica surface at the zero height, go to the Data Process'menu and select Flatten Base'in the Level'drop down menu.

Identify the extra cellular vesicles on the scanned surface by going to the Grains'menu and using Mark by Threshold'This algorithm identifies surface immobilized exosomes as particles protruding from the zero surface substrate by the height above the user selected threshold. Select a threshold in the range between one and three nanometers. This will eliminate most of the back ground interference.

Finally, perform geometric and dimensional characterization of the identified vesicles using the available distributions algorithms accessible from the Grains'menu. Export the AFM data from Gwyddion for specialized analysis by other computational tools and custom computer programs. The nickel chloride surface modification results in an immobilization of extra cellular vesicles that is time dependent.

The surface concentration of the immobilized vesicles is excessively dense after 24 hours of incubation whereas the 12 hour incubation leads to fewer exosomes and scan data that are easier to analyze accurately. This AFM image shows hydrated MCF7 exosomes electrostatically immobilized on the modified mica surface. The corresponding AFM phase image confirms that the grains in the height image are soft nano particles as should be expected for membrane vesicles.

The height data for three vesicles along the same line are shown here. These profiles illustrate a flattened shape caused by the electrostatic attraction of exosomes to the positively charge surface of the modified mica. The shape distortion is apparent in an enlarge view of the immobilized vesicle and its cross section.

To estimate the globular size of the exosomes in the solution, on can match the volumes enclosed by surface immobilized and spherical membrane envelopes. The size distribution of globular vesicles in the solution was determined from the AFM data of 561 immobilized vesicles. The vesicle sizes in CryoTEM images are consistent with the AFM results.

Before imaging the hyrdrated exosomes, it is important to remember to thoroughly rinse the surface with PBS. This will remove inbound exosomes and prevent their attachment to the AFM tip. When imaging the desiccated exosomes, be sure to use DI water to rise the substrate.

The DI wash will prevent the formation of salt crystals on the surface as the substrate dries. If present, salt crystals will make image processing a difficult task.