Comparison and development of scanning electron microscope techniques for delicate plant tissues

Area of Science:

• Plant Biology
• Microscopy Techniques
• Materials Science

Background:

• Cell deformation during scanning electron microscope (SEM) sample preparation affects result accuracy.
• Delicate samples, particularly plant tissues, are prone to deformation.

Purpose of the Study:

• To investigate the influence of various preparation methods on cell deformation in plant samples.
• To identify optimal SEM preparation techniques for minimizing artifacts in delicate tissues.

Main Methods:

• Classified plant samples as "delicate" based on water content and tissue hardness.
• Evaluated five preparation methods, measuring deformation at tissue and single-cell levels.
• Assessed sample stability and image quality under varying conditions (cryo-stage, voltage, sputter coating).

Main Results:

• Cryo- and methanol-fixation yielded minimal tissue deformation and superior cell shape preservation for delicate samples.
• Harder tissues were amenable to other dehydration-based preparation methods.
• Cryo-protectants and low cryo-stage temperatures (-30°C) enhanced stability and image quality.
• Larger sample size and cryo-stage application improved beam stability.

Conclusions:

• Cryo- and methanol-fixation are recommended for SEM preparation of delicate plant samples.
• Optimized sample preparation reduces artifacts and enhances SEM imaging reliability.
• Understanding preparation influences is crucial for accurate microscopic analysis.