Preparation of biological monolayers for producing high-resolution scanning electron micrographs

Area of Science:

• Materials Science
• Biotechnology
• Microscopy

Background:

• Scanning electron microscopy (SEM) is crucial for nanoscopic mapping of biological structures.
• Preparing biological samples for SEM, especially in vitro cultures, presents challenges in preserving cellular morphology and topography.
• High-resolution SEM (HR-SEM) requires samples to be conductive, thermally stable, and protected from structural alterations.

Purpose of the Study:

• To compare two coating methods (carbon vs. gold:palladium) for preparing biological samples for HR-SEM.
• To provide guidance on preparing in vitro epithelial or endothelial samples for HR-SEM.
• To evaluate the impact of coating on visualizing nanoscopic details of cellular membranes.

Main Methods:

• Comparative analysis of carbon and gold:palladium (Au:Pd) sputter coating techniques.
• Utilized Millicell cellulose inserts for culturing immortalized mouse brain endothelial cells (bEnd5).
• Examined topographical and membranous ultrastructures using HR-SEM.

Main Results:

• Au:Pd sputter coating produced micrographs with distinctly different topographical detail compared to carbon coating.
• Differences in nanoscopic detail resolution were observed between the two coating methods.
• Au:Pd coated samples yielded more descriptive imagery, aiding in profiling membrane nanostructures.

Conclusions:

• Au:Pd sputter coating is preferable to carbon coating for visualizing nanoscopic details of brain endothelial cell monolayers in HR-SEM.
• The choice of coating significantly impacts the quality and detail of micrographical data.
• Recommendations are provided for biological microscopists preparing tissue culture samples for HR-SEM.