A new technical approach for preparing frozen biological samples for electron microscopy

Area of Science:

• Plant biology
• Cryo-electron microscopy
• Cellular ultrastructure

Background:

• Limited knowledge exists on extracellular ice formation's impact on plant cellular ultrastructure.
• Current methods fail to maintain the cold chain during fixation for transmission electron microscopy (TEM).
• Chemical fixation is not feasible at sub-zero temperatures, and high-pressure freeze fixation (HPF) cannot freeze samples.

Purpose of the Study:

• To present a novel technical approach for preserving the cold chain of frozen plant samples before and during HPF.
• To enable the study of cellular ultrastructure consequences of extracellular ice formation in plants.

Main Methods:

• Development of an automatic freezing unit (AFU) from a standard laboratory freezer.
• Freezing of various algae and higher plant tissues at sub-zero temperatures (-2°C to -6°C).
• Transfer of frozen samples to HPF device and subsequent cryo-fixation without thawing, followed by cryo-substitution and TEM processing.

Main Results:

• Successful freezing and preparation of diverse plant samples using the AFU at sub-zero temperatures.
• Preceding tests confirmed that applied temperatures did not impair photosynthetic electron transport or cell vitality.
• TEM micrographs revealed excellent preservation of cellular ultrastructure, superior to ambient temperature fixation.

Conclusions:

• The presented method successfully preserves plant cell ultrastructure in a frozen state during cryo-fixation.
• High-quality TEM images provide a better understanding of extracellular ice formation's effects on cellular ultrastructure.
• This technique can offer new insights into organelle changes, intracellular injuries, and freezing/thawing processes, and can be combined with analytical TEM techniques.