The use of a simple method to avoid cell shrinkage during SEM preparation

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biochemistry

Background:

• Specimen preparation for scanning electron microscopy (SEM) often involves chemical fixation and dehydration processes.
• Critical point drying is a common method used to remove transitional fluid without causing surface tension damage.
• Standard fixation protocols can lead to significant cellular volume loss, impacting morphological accuracy.

Purpose of the Study:

• To investigate methods for reducing cellular shrinkage during specimen preparation for SEM.
• To evaluate the efficacy of additional chemical treatments in preserving cell volume.

Main Methods:

• Cells were fixed using glutaraldehyde and osmium tetroxide (OsO4).
• Specimens underwent critical point drying.
• Comparative analysis of cellular diameter was performed between standard fixation and a protocol including tannic acid and uranyl acetate treatments.

Main Results:

• Standard glutaraldehyde and OsO4 fixation followed by critical point drying resulted in a mean cellular diameter reduction of approximately 45%.
• Successive treatment with glutaraldehyde, OsO4, tannic acid, and uranyl acetate significantly reduced shrinkage to about 5%.

Conclusions:

• The addition of tannic acid and uranyl acetate to the fixation protocol is highly effective in preventing cellular shrinkage.
• This enhanced protocol improves the morphological fidelity of cells prepared for scanning electron microscopy.