Preparation of Hydrogels for Imaging by SEM

Protocol for Dehydration and Critical Point Drying (CPD) of Hydrogels for SEM

This protocol is intended for preserving the microstructure of hydrogels for conventional SEM imaging. Critical point drying minimizes surface-tension forces during drying and generally preserves structure better than air drying or freeze-drying.

Materials

• Hydrogel samples
• 0.1 M phosphate buffer (PBS) or appropriate buffer
• 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer
• Distilled water
• Ethanol solutions:
  • 30%
  • 50%
  • 70%
  • 90%
  • 95%
  • 100% (anhydrous)
• Critical point dryer (i.e., Leica EM CPD300)
• SEM stubs
• Conductive carbon tabs
• Sputter coater (Pt or Ir)

 

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Sample Fixation

1. Cut hydrogel into pieces approximately 2-5 mm thick.
2. Immerse samples in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer.
3. Fix for:
   • 2 hours or overnight at 4°C.
4. Rinse samples 3 × 10 minutes in buffer.

Optional Secondary Fixation

For hydrogels containing cells or extracellular matrix:

1. Incubate in 1% osmium tetroxide for 1 hour.
2. Wash 3 × 10 minutes with distilled water.

Note: Osmium tetroxide improves membrane contrast and structural stability but is often unnecessary for acellular hydrogels.

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Graded Ethanol Dehydration

Transfer samples sequentially through ethanol:ddH2O solutions:

Important Notes

• Use gentle agitation.
• Ensure complete immersion.
• Large or highly swollen hydrogels may require 30-60 minutes per step.
• Slow dehydration minimizes shrinkage and cracking.

 

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Critical Point Drying

CO₂ Exchange

1. Place samples in the CPD chamber of the automatic Leica CPD300 critical point dryer while submerged in 100% ethanol.
2. Perform multiple ethanol-to-liquid CO₂ exchanges.
3. Continue exchanges until ethanol has been completely replaced by liquid CO₂.

Typical settings:

• 10-15 exchange cycles
• Chamber temperature 8-15°C
• Pressure according to instrument specifications

Critical Point Transition

1. Raise chamber temperature and pressure above the critical point of CO₂:
   • Critical temperature: 31.1°C
   • Critical pressure: 7.38 MPa (1070 psi)
2. Hold for 10-15 minutes.
3. Slowly vent the chamber according to the instrument protocol.

Rapid venting can cause sample damage.

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Mounting

1. Mount dried specimens on aluminum SEM stubs using conductive carbon tabs.
2. Orient fracture surfaces upward if you plan to examine internal morphology.

Cross-Section Preparation

For internal pore analysis:

• Mechanically fracture the hydrogel after CPD.
• Alternatively, use PFIB milling or FIB cross-sectioning for cleaner internal views.

 

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Sputter Coating

Use the Leica EM ACE600 sputter coater to apply a conductive coating:

Iridium is preferred when imaging nanoscale features because it produces finer grain size.

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Quanta 450 FE-SEM Imaging Conditions

Suggested starting conditions:

Lower accelerating voltages generally provide better surface detail and reduce charging.

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Common Artifacts