Scanning Electron Microscopy

Abstract

AbstractScanning 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 SEMAlternate Protocol 1: Practical considerations for the preparation of soft tissuesAlternate Protocol 2: Removal of debris from the exoskeleton of invertebratesAlternate Protocol 3: Fixation of colonies grown on agar platesAlternate Protocol 4: Stabilization of polysaccharide structures with alcian blue and lysineAlternate Protocol 5: Preparation of non‐adherent particulates in solution for SEMSupport Protocol 1: Application of thin layer of adhesive on substrate to improve adherenceSupport Protocol 2: Poly‐L‐lysine coating specimen substrates for improved adherenceSupport Protocol 3: Microwave processing of biological specimens for examination by SEMBasic Protocol 2: Critical point drying of specimensAlternate Protocol 6: Chemical alternative to critical point dryingBasic Protocol 3: Sputter coatingAlternate Protocol 7: Improved bulk conductivity through “OTOTO”Basic Protocol 4: Immune‐labeling strategiesAlternate Protocol 8: Immune‐labeling internal antigens with small gold probesAlternate protocol 9: Quantum dot or fluoronanogold preparations for correlative techniquesBasic Protocol 5: Exposure of internal structures by mechanical fracturingBasic Protocol 6: Exposure of internal structures of tissues by fracturing with liquid nitrogenBasic Protocol 7: Anaglyph production from stereo pairs to produce 3D images