Scanning Electron Microscopy

Abstract

Scanning electron microscopy (SEM) remains distinct in its ability to allow topographical visualization of structures. Key elements to consider for successful examination of biological specimens include appropriate preparative and imaging techniques. Chemical processing induces structural artifacts during specimen preparation, and several factors need to be considered when selecting fixation protocols to reduce these effects while retaining structures of interest. Particular care for proper dehydration of specimens is essential to minimize shrinkage and is necessary for placement under the high-vacuum environment required for routine operation of standard SEMs. Choice of substrate for mounting and coating specimens can reduce artifacts known as charging, and a basic understanding of microscope settings can optimize parameters to achieve desired results. This article describes fundamental techniques and tips for routine specimen preparation for a variety of biological specimens, preservation of labile or fragile structures, immune-labeling strategies, and microscope imaging parameters for optimal examination by SEM. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Chemical preparative techniques for preservation of biological specimens for examination by SEM

Alternate Protocol 1: Practical considerations for the preparation of soft tissues

Alternate Protocol 2: Removal of debris from the exoskeleton of invertebrates

Alternate Protocol 3: Fixation of colonies grown on agar plates

Alternate Protocol 4: Stabilization of polysaccharide structures with alcian blue and lysine

Alternate Protocol 5: Preparation of non-adherent particulates in solution for SEM

Support Protocol 1: Application of thin layer of adhesive on substrate to improve adherence

Support Protocol 2: Poly-L-lysine coating specimen substrates for improved adherence

Support Protocol 3: Microwave processing of biological specimens for examination by SEM

Basic Protocol 2: Critical point drying of specimens

Alternate Protocol 6: Chemical alternative to critical point drying

Basic Protocol 3: Sputter coating

Alternate Protocol 7: Improved bulk conductivity through “OTOTO”

Basic Protocol 4: Immune-labeling strategies

Alternate Protocol 8: Immune-labeling internal antigens with small gold probes

Alternate protocol 9: Quantum dot or fluoronanogold preparations for correlative techniques

Basic Protocol 5: Exposure of internal structures by mechanical fracturing

Basic Protocol 6: Exposure of internal structures of tissues by fracturing with liquid nitrogen

Basic Protocol 7: Anaglyph production from stereo pairs to produce 3D images