High-resolution scanning electron microscopy of frozen-hydrated cells

Area of Science:

• Electron Microscopy
• Cell Biology
• Biophysics

Background:

• Low-temperature scanning electron microscopy (LTSEM) is crucial for visualizing biological structures.
• Optimizing sample preparation is key to achieving high-resolution imaging in cryo-electron microscopy.

Purpose of the Study:

• To develop and investigate sample processing techniques for LTSEM.
• To evaluate imaging parameters for frozen-hydrated and partially dehydrated biological specimens.

Main Methods:

• Investigation of cryo-fixed yeast, Paramecia, and sea urchin embryos using a field-emission scanning electron microscope with a cold stage.
• Imaging of uncoated frozen-hydrated samples with low-voltage backscattered electron (BSE) signals.
• Comparison of imaging results between uncoated and platinum cryo-coated samples.

Main Results:

• Sufficient resolution and contrast were achieved for visualizing cellular structures like membranes and nuclear pores in uncoated frozen-hydrated samples.
• Platinum cryo-coating enhanced resolution, enabling visualization of intramembranous particles.
• Partially dehydrated samples showed potential for improved imaging with reduced electron beam-induced mass loss and fewer artifacts.

Conclusions:

• LTSEM of uncoated, frozen-hydrated samples can reveal fine cellular details, limited by backscattered electron detection sensitivity at low accelerating voltages.
• High-resolution imaging of intramembranous particles is feasible with cryo-coated specimens.
• Partially dehydrated samples represent a promising approach for cell biology, offering advantages over chemical fixation and reducing beam damage.