This article describes a method for visualizing extracellular vesicles (EVs) secreted by cyanobacteria using transmission electron microscopy (TEM). The process involves preparing a TEM grid, applying a suspension of EVs, and staining them to enhance imaging contrast.
Place a polyvinyl formal resin-coated transmission electron microscopy or TEM grid on a clean plastic film.
Add a suspension of concentrated and purified extracellular vesicles or EVs, nanosized, membrane-bound particles, onto the grid.
These EVs are secreted by cyanobacteria and carry macromolecules within their membranes.
Incubate the grid to allow the EVs to adsorb onto its resin-coated surface.
Touch the grid edge with a clean filter paper to absorb the excess suspension.
Float the grid on uranyl acetate to stain the adhered vesicles.
Uranyl acetate surrounds the vesicles, creating a dark outline that enhances contrast during imaging.
Remove the grid and use filter paper to absorb the excess stain.
Wash the grid with ultrapure water to remove any residual stain. Repeat the wash and then allow it to air dry.
Finally, visualize the EVs using TEM to assess their morphology, size, and purity.
Carefully apply five microliters of vesicle and let it sit for five minutes. Remove the sample by touching the edge of a grid to a piece of clean filter paper.
Pipette out 20 to 50 microliters of 2%uranyl acetate onto a flat surface covered by plastic film. Then place the grid floating on top of it for two minutes. Remove uranyl acetate using filter paper and float briefly on a drop of ultrapure water to wash.
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