Scanning Electron Microscopy
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 unit 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. Curr. Protoc. Microbiol. 25:2B.2.1‐2B.2.47. © 2012 by John Wiley & Sons, Inc.
Keywords: scanning electron microscopy; immune‐labeling; EM specimen preparation; critical‐point drying; sputter coating; specimen fracturing; microwave‐processing; cryo‐SEM quantum dots
Table of Contents
- Safety Considerations
- 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 Tiissues
- 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 Conventional SEM
- Basic Protocol 2: Critical‐Point Drying 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 Preparation for Correlative Techniques
- Basic Protocol 5: Exposure of Internal Structures by Mechanical Fracturing
- Basic Protocol 6: Anaglyph Production from Stereo Pairs to Produce 3‐D Images
- Reagents and Solutions
- Literature Cited
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 Tiissues
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 Conventional SEM
Basic Protocol 2: Critical‐Point Drying 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 Preparation for Correlative Techniques
Basic Protocol 5: Exposure of Internal Structures by Mechanical Fracturing
Figure 2.B0.1 Thermanox or aclar materials can easily be trimmed or punched to desired dimensions using scissors or punch tool for adaptation to size requirements for subsequent processing steps.
Figure 2.B0.2 (A) Removal of prescored silicon wafer followed by application of specimen in suspension (B) and, (C) proper dispersal of fluids.
Figure 2.B0.3 Low‐magnification SEM image of Ornithodoros hermsi tick (A), and higher magnification images of uncleaned (B, D, E) compared with cleaned tick exoskeleton (C, F). Scale bars as indicated.
Figure 2.B0.5 Agarose bead with dendrocytes adhered to silicon chip precoated with thin layer adhesive. Scale bar = 25 µm.
Figure 2.B0.6 Placement of a chip in specimen vessel used in a Bal‐Tec CPD.
Figure 2.B0.7 Membrane filter insert removed from a 24‐well tissue culture plate (A) is quickly transferred and immersed for trimming (B) and the membrane is removed with fine‐tipped forceps (C) for placement in CPD container.
Figure 2.B0.8 Carefully place the lid on the specimen chamber and make sure it is properly threaded and closed tightly as this chamber reaches high pressure.
Figure 2.B0.9 HeLa cells grown on silicon chips were dehydrated using either a CPD (A) or HMDS (B). Specimens were coated with 80 Å of Ir. Scale bar = 1 µm.
Figure 2.B0.10 Double‐sided adhesive tab placeed on aluminum SEM stub (A) and removal of protective layer with fine‐tipped forceps (B).
Figure 2.B0.12 Conductive paint is carefully applied with a brush to form complete contact with the underlying surface. If the substrate hangs slightly over the stub, extra paint can be applied on the bottom side of the coverslip.
Figure 2.B0.13 Silver paint was carefully applied after sputter‐coating tissues mounted on prepared SEM stubs to improve contact between tissue and conductive surface.
Figure 2.B0.15 Chlamydia‐infected HeLa cells were prepared by OTOTO and left uncoated for examination (A, B), minimally coated with 20 Å of Cr, (C, D) or conventionally processed and coated with 75 Å of Ir (E, F). Scale bars = 0.5 µm.
Figure 2.B0.16 Yersinia pestis labeled with 20 nm gold, imaged at a nominal magnification of 35,000× (A) compared with Staphylococcus epidermidis labeled with 10 nm gold and visualized at a nominal magnification of 60,000× (B). Scale bars = 0.5 µm.
Figure 2.B0.17 Bacteria in ovarian tissue labeled with Nanogold and silver enhanced for 15 min to improve visualization by SEM. Scale bar = 0.5 µm.
Figure 2.B0.19 Scotch tape is gently applied to cell layer grown on Thermanox coverslips.
Figure 2.B0.21 Aligned stereo pair.
Figure 2.B0.22 Images converted to RGB mode.
Figure 2.B0.23 Red channel removed from left image.
Figure 2.B0.24 Red image from right image copied on to left image.
Figure 2.B0.25 Selection of the RGB channel shows overlay of the new red channel, creating an anaglyph for 3‐D viewing using red/blue glasses.
Figure 2.B0.26 Chlamydia‐infected HeLa cells high‐pressure frozen and freeze fractured after fixing with PFA only (A) or with 2.5% GA/0.1% MG and OTOTO treatment (B). Scale bar = 5 µm.
Figure 2.B0.27 Polarized epithelial cells grown on membrane filters after drying and coating, revealed damage to the monolayer (A) and at higher magnification damage to the membrane was evident (B). Scale bar = 0.5 µm.
Figure 2.B0.29 Immune‐labeled bacteria demonstrating excessive background labeling in the out‐of‐focus regions (A), moderate background labeling (B), or minimal levels of background labeling (C). Scale bars = 0.5 µm.
Figure 2.B0.30 Immune‐labeled Staphylococcus epidermidis prepared by conventional EM techniques demonstrates detachment of structure of interest. Scale bar = 0.25 µm.
Figure 2.B0.31 Chlamydia‐infected HeLa cells were immune‐labeled intracellularly for antigens against a bacterial surface protein using Nanogold followed by silver enhancement and viewed by either cryo‐SEM (A) or TEM (B). Scale bar = 0.5 µm.
Figure 2.B0.32 Macrophages grown on aclar coverslips, conventionally prepared and coated with 75 Å of Ir were imaged by SEM at 2 kV (A), 5 kV (B), or 10 kV (C). Scale bar = 1 µm.
Figure 2.B0.34 HIV‐infected T lymphocyte imaged with 5 kV at a working distance of 5 mm (A) or 17 mm (B). Scale bar = 1 µm.
Figure 2.B0.35 Images of a macrophage show astigmatism in the x direction (A), y direction (B), corrected (C), corrected and focused (D). Scale bar = 2 µm.
Figure 2.B0.37 HeLa cell co‐infected with VZV and Streptococcus. Scale bar = 1.5 µm.
Figure 2.B0.38 Staphylococcus epidermidis immune‐labeled for biofilm localization shown in both SE (A) and mixed SE and BSE (B) imaging mode to allow visualization of gold particles. Scale bar = 0.5 µm.
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|Palade, G.E. 1951. A study of fixation for electron microscopy. J. Exp. Med. 95:285‐297.|
|Sabatini, D.D., Bensch, K., and Barrnett, R.J. 1963. Cytochemistry and electron microscopy: The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell. Biol. 17:19‐58.|
|Glauert, A.M. 1974. Fixation, Dehydration, and Embedding of Biological Specimens. Elsevier, Amsterdam.|
|The following books are excellent resources for general EM preparative techniques covering general principle for conventional and immunological preparation.|
|Hayat, M.A. 1989. Colloidal gold: Principles, Methods, and Applications. Volumes 1‐3. Academic Press, San Diego.|
|Hayat, M.A. 2000. Principles and Techniques of Electron Microscopy: Biological Applications. Cambridge University Press, New York.|
|The above Web sites are some common sources for electron microscopy supplies, reagents and useful tips for immune‐labeling and microwave oven processing.|
|M. Sanders at the University of Minnesota. A University site containing particularly useful microwave oven theory and protocols.|
|Advanced Microscopy Facility, University of Victoria. A University site containing particularly useful microwave oven theory and protocols.|
|Above are additional resources for commercially available antibodies and small gold probes|
|Anaglyph method from Bob Mannle's Web site, Micro Format.|