TEM Tips for Imaging Uranyl Acetate–Negative–Stained Particles

(Liposomes, extracellular vesicles, viruses, protein–lipid assemblies, etc.)

1. Grid Preparation

• Use carbon‑coated 200-mesh copper grids (Formvar‑supported carbon if your vesicles are very delicate).
• Glow discharge the grids ~20–30 seconds before use.
  • This makes the carbon film hydrophilic, improving even particle spreading and preventing beading.

Tip: Poor wetting = uneven staining + distorted vesicles.

2. Sample Application

• Apply 3–5 µL of sample to the grid.
• Allow 30–60 seconds for adsorption.
• Avoid highly concentrated samples; dilute to ~10⁸–10⁹ particles/mL for clean, non‑overlapping fields.

Tip: Too many particles cause clumping, flattening, and staining artifacts.

3. Washing Step (Optional but recommended for EVs)

• After blotting the sample, briefly touch the grid to a small drop of filtered water.
  • This removes salts that can crystallize and obscure fine detail.

Tip: One quick wash is usually enough—too much washing may dislodge vesicles.

4. Uranyl Acetate (UA) Staining

• Use 1–2% aqueous uranyl acetate, freshly filtered (0.02 μm syringe filter).
• Apply 3–5 µL to the grid for 10–30 seconds, depending on particle type.
• Blot gently with filter paper from the grid edge.

Avoid common UA artifacts:

• Dry stain crystals → indicate slow or uneven blotting.
• Stain pooling → suggests a hydrophobic grid or too much residual buffer.
• Overstaining → causes high contrast but loss of membrane definition.

Tip: For delicate EVs, a short 10–15 s stain often gives the most natural morphology.

5. Particle Morphology Considerations

Liposomes

• Negative stain may flatten spherical liposomes—this is normal.
• Expect “cup‑shaped” deformation due to dehydration forces.
• Multi‑lamellar structures may appear as onion‑ring shells in stain.

Extracellular Vesicles (EVs)

• EVs often appear as round or cup‑shaped, depending on fixation and drying.
• The presence of protein contaminants may appear as small dark granules around vesicles.
• If vesicles appear collapsed, lower their adsorption time or apply stain earlier.

6. Grid Drying

• Allow grids to air‑dry completely, ideally ≥ 5–10 minutes, or use a heat lamp.
• Keep grids protected from dust (e.g., inside a covered petri dish).

Tip: Residual moisture produces image drift and charging.

7. Imaging Conditions

• Use low-dose TEM, if available (soft biological material is beam‑sensitive).
• Recommended initial settings:
  • 80–120 kV acceleration voltage
  • Low‑dose mode if available
  • Minimal beam exposure when searching for areas
• Start at lower magnification (5,000–20,000×) to locate vesicles, then move to 50,000–120,000× for details.

Tip: Excessive beam can cause vesicle collapse, UA bubbling, and membrane tearing.

8. Avoiding Common Negative Stain Problems

Problem: "Doughnut" or “halo” appearances

Cause: Stain excluded from the center due to lipid content.
Fix: Try a shorter adsorption time or a very brief pre‑stain wash.

Problem: Crystals everywhere

Cause: Salt from sample buffer.
Fix: Add a quick distilled water dip before staining.

Problem: Vesicle rupture

Cause: Harsh blotting or highly charged grids.
Fix:

• Blot more gently
• Reduce glow discharge time
• Lower sample adsorption time

9. Sample Buffer Recommendations

• Avoid buffers high in salts or divalent cations (e.g., PBS, HEPES with Mg²⁺ or Ca²⁺).
• Use low‑salt buffers when possible (e.g., 10 mM Tris).
• Avoid detergents—these dissolve vesicle membranes.

10. Quantification & Image Quality Considerations

• Negative staining is excellent for morphology but unreliable for size quantification due to dehydration artifacts.
• Use cryo‑TEM or NTA (nanoparticle tracking analysis) for accurate size distributions.