1. Before Imaging
Check Grid Quality
- Inspect grids under a light microscope if possible, look for:
- Tears in the carbon film
- Crystals or debris (often salts from insufficient washing)
- Stain puddles indicate poor drying
- Always handle grids with clean, anti‑capillary tweezers.
Confirm Stain Quality
- With uranyl acetate staining:
- Good grids show a uniform light‑gray tone.
- Dark pools or crystals indicate inadequate rinsing or drying.
- If the stain is too light, increase the stain time slightly (1.5–2 min).
2. Microscope Setup
Initial Settings
- Accelerating voltage:
- Typically, 120 kV for biological samples.
- Higher keV improves penetration but may reduce contrast.
- Spot size:
- Start with spot size 2 for a balance of brightness and dose.
- Condenser aperture:
- Smaller apertures = more contrast but lower brightness.
- For immunogold: use a small or medium condenser aperture to enhance gold visibility.
Objective Aperture
- In immunogold imaging, inserting a small objective aperture (e.g., 40–60 µm):
- Enhances contrast
- Accentuates electron‑dense structures (gold particles)
3. Focusing and Stigmation
Proper Focusing Techniques
- Focus at a moderate magnification (e.g., 10–20k×), then zoom in.
- Avoid focusing at very high magnification initially—noise dominates.
Astigmatism Correction
- Gold particles reveal astigmatism easily (appear elongated or “egg‑shaped”).
- Use objective stigmators to make gold particles perfectly round.
4. Imaging Immunogold Labelling
Gold Particle Identification
- 5–20 nm gold particles appear as:
- Black, perfectly round dots
- Uniform size (depending on the secondary antibody conjugate)
Check for Aggregation
- Aggregates appear as particle clusters—this may indicate:
- Old antibody
- Contaminated solutions
- Improper handling (vortexing damaged conjugates)
Magnifications
- Survey tissue at low/intermediate magnification:
- 1,500×–4,000× for overview
- 8,000×–20,000× for identifying regions of interest
- For gold localization:
- 25,000×–80,000× depending on particle size
- 10 nm gold is typically best imaged around 40k×–60k×
5. Contrast Optimization
Biological Contrast
- Biological samples are low‑contrast; adjust:
- Brightness/contrast
- Objective aperture size
- Defocus (slightly underfocus for phase contrast)
Underfocus Strategy
- Underfocusing by −1 to −2 µm increases visibility of membranes and gold particles.
- Use careful judgment—too much underfocus lowers resolution.
6. Electron Dose Management
Minimize Beam Damage
- Biological grids are beam‑sensitive; avoid prolonged exposure.
- Use low‑dose mode if available.
- Move to a fresh area frequently to prevent bubbling or darkening.
Imaging Strategy
- Focus on a nearby, unimportant area, then move to your region of interest to capture the final image.
7. Drift and Stability
Let the Grid Settle
- Allow the holder to equilibrate for 5–10 minutes after insertion before fine imaging.
- Thermal drift will decrease significantly.
Mechanical Stability
- Avoid touching the stage or column during imaging.
- If drift persists:
- Increase magnification slowly
- Use software drift correction (if available)
8. Capturing High‑Quality Images
Camera Settings (CCD/CMOS)
- Choose a binning level appropriate for your resolution needs.
- Lower binning = higher resolution, more noise
- Higher binning = smoother images, lower resolution
- Longer exposure time increases contrast but can cause drift—balance carefully.
Image Composition
- Include:
- Clear membrane structures
- Multiple gold particles for quantification
- Scale bar automatically added by the software
9. Recognizing Common Problems
If everything looks washed out:
- Increase underfocus
- Use a smaller objective aperture
- Increase exposure time
If the image is too dark or noisy:
- Increase beam intensity slightly
- Increase binning
- Use auto‑contrast/auto‑brightness functions sparingly
If gold particles are hard to see:
- Adjust brightness/contrast
- Insert a smaller objective aperture
- Confirm that the stain is not too thick
10. After Imaging
Grid Storage
- Store grids in labelled TEM grid boxes:
- At room temperature
- In a dust‑free drawer or desiccator
- Avoid handling the same grid excessively; mechanical damage accumulates.