Sectioning, Staining and TEM Imaging of Embedded Exosomes: A Protocol To Visualize the Structural Features of Exosomes Using TEM

Exosomes are nanosized, spherical vesicles enclosed by a lipid bilayer containing DNA, RNA, and protein molecules present inside the lumen.

To visualize the exosome structure using transmission electron microscopy, or TEM, begin by taking an exosome block - a solid matrix block containing the embedded exosomes. Using an ultra-microtome, obtain ultrathin sections of the exosome block.

Collect the exosome section on a suitable grid and allow it to adhere to the surface. The grid provides stable support to the exosome specimen for subsequent staining steps.

Next, position the grid over a drop of uranyl acetate - a heavy metal stain - and incubate for the desired duration. Uranyl ions interact with lipids, proteins, DNAs, and RNAs present in the exosomes.

Subsequently, stain the exosome section with lead citrate, which also binds to the exosomal biomolecules. Lead citrate weakly binds to the pre-bound uranyl ions and enhances the contrast against background during imaging.

Observe the doubly stained exosomes using TEM. TEM focuses the electron beam on the exosome sample. Stained regions in the exosome scatter electrons more strongly in comparison to the unstained regions.

The objective aperture filters these highly scattered electrons. Thus, exosomes containing stained biomolecules appear darker than the background.

Using an ultra-microtome, prepare sections with a thickness of 60 nanometers. Place the sections on a nickel grid and double stain some of them with 2% uranyl acetate for 20 minutes, followed by lead citrate for 10 minutes. Next, place the stained section into a transmission electron microscope and view the sample using 80 kV and follow automatic settings for the exposure time. With an exosome image in view, acquire and save the image using the microscope's software.