A simple pneumatic device for plunge-freezing cells grown on electron microscopy grids

Area of Science:

• Cryo-EM sample preparation
• Cell biology
• Biophysics

Background:

• Rapid freezing is crucial for preserving cellular structures in cryo-electron microscopy (cryo-EM).
• Conventional plunge-freezing methods can be complex and expensive.
• Maintaining sample integrity during transfer to cryo-fixation or substitution media is challenging.

Purpose of the Study:

• To present a detailed design for a simple, inexpensive, variable-speed pneumatic plunge-freezing device.
• To optimize the plunge-freezing process for cultured cells.
• To ensure sample integrity during transfer to subsequent cryo-preservation steps.

Main Methods:

• A pneumatic device was designed to drive cultured cells on gold grids into a propane/isopentane mixture cooled by liquid nitrogen.
• A vacuum system was employed to manage cryogenic vapors and prevent premature freezing.
• A compressed air system was used to create a sub-zero environment for sample transfer.

Main Results:

• The device achieves variable speeds from 1.0 to 5.8 m/s for rapid freezing.
• The vacuum system effectively prevents sample freezing in cryogenic vapors.
• The compressed air system maintains temperatures below -120°C for sample transfer, preventing thawing.

Conclusions:

• The described pneumatic plunge-freezing device is a cost-effective and efficient tool for high-quality cryo-EM sample preparation.
• The design addresses key challenges in rapid freezing and sample transfer, enhancing cryo-preservation.
• This method facilitates the study of cellular structures at high resolution using cryo-EM.