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Quanta 450 ESEM – Step‑by‑Step Protocol for Imaging Hydrogels

Quanta 450 ESEM – Step‑by‑Step Protocol for Imaging Hydrogels

The Thermo Fisher / FEI Quanta 450 ESEM supports three vacuum modes—high vacuum, low vacuum, and ESEM mode using water vapour. Hydrogels should be imaged in ESEM mode to maintain hydration and prevent structural collapse.

This protocol incorporates the validated ESEM principles described in Thermo Fisher’s application note on hydrated samples and peer‑reviewed hydrogel imaging studies.

1. Sample Preparation (Minimal)

1.1. Trim and handle the hydrogel

  1. Cut a small piece that fits on the Quanta stub.
  2. Handle gently to avoid deformation—hydrated gels are mechanically weak and easily distorted.
    • ESEM imaging is intended to avoid dehydration‑induced artifacts common in hydrogels.

1.2. Mount onto Quanta 450 stub

  1. Place the hydrogel directly on a flat stub.
  2. Select the Peltier cooling stage, since temperature control helps retain hydration while avoiding ice formation.
    • Cooling slows evaporation without freezing, preventing ice‑crystal damage.

Note: Do not sputter‑coat. No conductive coating is required for ESEM mode.

2. Chamber Loading & ESEM Configuration

2.1. Load into the Quanta chamber

  1. Insert the stub into the chamber and center it over the stage.
  2. Select ESEM Mode on the Quanta user interface before pumping down.

2.2. Select water vapour as imaging gas

  1. The Quanta 450 automatically controls water-vapour injection. Choose H₂O vapour from the ESEM gas selection menu.
    • Using pure water vapour (instead of moist air) ensures stable, predictable humidity control.

3. Establishing Stable Hydration Conditions

3.1. Set stage temperature

  1. Start around 2–5 °C using the Peltier stage controls.
    • Cooling maintains hydration and prevents premature evaporation. Freezing must be avoided.

3.2. Increase chamber pressure

  1. Slowly increase pressure to the water-vapour equilibrium region (varies slightly depending on stage temperature; Quanta will display the vapour pressure curve).
  2. Watch for:
  • Too much condensation → surface becomes obscured.
  • Too little humidity → surface begins drying.
  • The goal is a stable, visible, hydrated surface.

3.3. Fine‑tune equilibrium

  1. Adjust the temperature ±1 °C and the pressure in small increments until condensation disappears without drying.
  2. Once stabilized, ESEM mode can hold equilibrium for hours.

4. Imaging on the Quanta 450

4.1. Set imaging parameters

  1. Begin with:
  • Low accelerating voltage (5–10 kV) to minimize beam damage.
  • Spot size: Medium–small (appropriate for hydrated, non‑conductive samples).
  • Detector: Use the Gaseous Secondary Electron (GSE) detector for ESEM.
  1. Focus slowly; the electron‑gas interactions in ESEM can make focusing less sharp than in high vacuum.

4.2. Capture hydrated-state microstructure

  1. Acquire images of the fully hydrated surface.
  • High humidity preserves native morphology but reduces feature sharpness.

5. Optional: Controlled Drying Imaging Series

Useful for revealing fibre networks, pore structures, or microarchitecture.

5.1. Gradually reduce chamber pressure

  1. Lower the pressure slowly (e.g., steps of 20–40 Pa) to evaporate a small amount of water.
  • Partial drying reveals internal structures such as collagen fibres.

5.2. Capture images at each hydration level

  1. Document morphology at:
  • Fully hydrated
  • Slightly dehydrated
  • Semi‑dry
  • Nearly dry

ESEM hydrogel galleries show how structures change during swelling/drying cycles. (https://polymermicroscopy.com/eng_sem_hydro.htm)

Caution: Over‑drying can cause weakly crosslinked hydrogels to collapse.

6. Ending the Session

6.1. Return to safe conditions

  1. Restore humidity and stage temperature before venting.

6.2. Vent and remove sample

  1. Vent the chamber normally via the Quanta interface, then remove the sample.

Summary – Quanta 450‑Specific Highlights

  • Use ESEM Mode with H₂O vapour (Quanta auto‑controls vapour pressure).
  • The Peltier cooling stage is highly beneficial for hydrogel stability.
  • The GSE detector is the default for hydrated samples in ESEM.
  • Fine control of pressure & stage temperature is essential for hydrated equilibrium.
  • Imaging a hydration series is extremely useful for hydrogel microstructure analysis.
  • All hydration‑control principles come from validated ESEM behaviour in hydrated materials.
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