High-pressure freezing of mechanically stretched cells

Area of Science:

• Electron Microscopy
• Cell Biology
• Biophysics

Background:

• High-pressure freezing (HPF) is a crucial electron microscopy (EM) technique for preserving cellular ultrastructure.
• Conventional HPF requires cells cultured on rigid sapphire discs.
• Cell stretch experiments necessitate the use of extensible growth supports like silicone membranes.

Purpose of the Study:

• To adapt HPF for use with elastic silicone membranes as a cell growth support.
• To evaluate the ultrastructural preservation of cells immobilized on silicone membranes using HPF.
• To enable HPF preparation for cells undergoing mechanical stress experiments.

Main Methods:

• Developed a method to clamp stretched silicone membranes for HPF apparatus compatibility.
• Performed HPF on cells cultured on silicone membranes.
• Compared ultrastructural preservation with conventional HPF on sapphire discs and chemical fixation.

Main Results:

• HPF immobilization on silicone membranes yielded improved structural preservation compared to chemical fixation.
• Ultrastructural quality was lower than HPF on sapphire discs.
• Increased freezing artifacts were observed in silicone membranes, likely due to reduced heat transfer.

Conclusions:

• HPF immobilization is feasible with growth supports beyond sapphire discs, including stretched silicone membranes.
• This technique broadens the applicability of HPF for studying cells on unconventional substrates.
• Further optimization may be needed to mitigate freezing artifacts on silicone membranes.