Visualization of Cyanobacterial Extracellular Vesicles Using Transmission Electron Microscopy

Abstract

Source: Biller, S. J., et al. Isolation and Characterization of Cyanobacterial Extracellular Vesicles. J. Vis. Exp. (2022)

This video demonstrates the preparation of cyanobacterial extracellular vesicles for transmission electron microscopy by negative staining, enabling high-resolution visualization of vesicle morphology, size, and purity for structural and comparative analyses.

Protocol

1. Characterization of the isolated vesicles

1. Perform negative stain transmission electron microscopy (see Table of Materials) (TEM - transmission electron microscopy; particle size, structure, purity).
   1. To improve the image quality, glow discharge the surface of a former-coated TEM grid using a glow discharge system according to the manufacturer's guidelines (see Table of Materials).
   2. Carefully apply ~5 µL of the vesicle sample and let sit for 5 min.
      NOTE: Samples with vesicle concentrations >10⁹ mL^-1 will typically give the best results; more dilute samples will have few vesicles per image.
   3. Remove the sample by touching the edge of a grid to a piece of clean filter paper.
   4. Pipette a 20-50 µL drop of 2% uranyl acetate (see Table of Materials) onto a flat surface covered by plastic film, and place the grid floating on top of it for 2 min.
   5. Remove uranyl acetate using filter paper and float briefly on a drop of ultrapure water to wash. Repeat the water wash a second time.
      NOTE: The final dry grid is ready for visualization after step 1.1.5.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2% Uranyl acetate solution	Electron Microscopy Sciences	22400-2	Negative staining - TEM
Carbon Support Film 200 Mesh, copper. CF200-Cu	Electron Microscopy Sciences	71150	Transmission electron microscopy (TEM) grid
easiGlow Glow Discharge Cleaning System	Ted Pella	91000	Commercial TEM grid glow discharger
Milli-Q Reference Water Purification System	Merck	Z00QSV0WW	Water purification system for obtaining type I ultrapure grade water
Transmission electron microscope	JEOL	JEM-1400	Or similar microscope