Embedding media for cryomicrotomy: an applicative reappraisal

Area of Science:

• Cryo-electron microscopy and histology sample preparation.

Background:

• Cryoembedding media are crucial for high-resolution cryosectioning.
• Recent advancements in cryomicrotomes necessitate re-evaluation of embedding media formulations.

Purpose of the Study:

• To reassess and optimize cryoembedding media formulations for improved cryosectioning.
• To investigate the impact of polymer concentration and type on section quality.

Main Methods:

• Formulation of water-based polyvinyl alcohol (PVA) solutions with varying molecular weights (MW 195000 and 47000).
• Addition of polyethylene glycol (MW 380-420) to PVA solutions.
• Systematic testing of media across a range of polymer concentrations.
• Application of a wide temperature differential between specimen and knife (-11°C to -13°C vs. -33°C to -35°C).

Main Results:

• Identified critical polymer concentrations for optimal sectioning, avoiding wrinkles and folds.
• Polyethylene glycol addition significantly improved sectioning quality across a 2-100 micrometer range.
• Achieved reproducible serial sectioning at 2-micrometer intervals using disposable blades.
• Developed low and high viscosity media for precise sample orientation and sequential infiltration.

Conclusions:

• Optimized PVA-based cryoembedding media formulations facilitate superior cryosectioning.
• The developed media enable precise, reproducible serial sectioning for advanced microscopy techniques.
• This research provides improved cryoembedding solutions for cryomicrotomy applications.