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 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.

Figure 1

Thermanox™ or aclar materials can easily be trimmed or punched to desired dimensions using a scissors or punch tool for adaptation to size requirements for subsequent processing steps.

Figure 2

(A) Removal of pre-scored silicon wafer followed by application of specimen in suspension (B) and, (C) proper dispersal of fluids.

Figure 3

Low magnification SEM image of Ornithodoros hermsi tick (A), and higher magnification images of un-cleaned (B, D, & E) compared with cleaned tick exoskeleton (C & F). Scale bars as indicated.

Figure 4

SEM of Hms+ and Hms− Yersinia pestis grown as bacterial lawns on agar plates. The Hms+ colonies showed retention of an extracellular substance (A), while the mutant treated with the same alcian blue-lysine fixative mixture did not (B). Scale bars = 0.5 μm.

Figure 5

Agarose bead with dendrocytes adhered to silicon chip pre-coated with thin layer adhesive. Scale bar = 25 μm.

Figure 6

Placement of a chip in specimen vessel used in a Bal-Tec CPD.

Figure 7

Membrane filter insert removed from 24 cell tissue culture plate (A) is quickly transferred and immersed for trimming in (B) and the membrane is removed with a fine-tipped tweezers (C) for placement in CPD container.

Figure 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 9

HeLa cells grown on silicon chips were dehydrated using either a CPD (A) or HMDS (B). Specimens were coated with 80 Angstroms of Ir. Scale bar = 1 μm.

Figure 10

Double-sided adhesive tab place on aluminum SEM stub (A) and removal of protective layer with fine-tipped forceps (B).

Figure 11

Transwell membrane filters curl up into a scroll after drying in a CPD (A). After placing on the adhesive, it can simply be rolled across the tape (cell layer rolls inward) (B) and gently apply pressure for improved contact. Silver paint can be CAREFULLY applied to increase contact of membrane to adhesive (C).

Figure 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 13

Silver paint was carefully applied after sputter-coating tissues mounted on prepared SEM stubs to improve contact between tissue and conductive surface.

Figure 14

Specimen appearance after coating with 75 angstroms of Ir by visual or SEM examination of Salmonella infected polarized HeLa cells grown on transwell membrane filters (A, B), non-adherent red blood cells infected with malarial parasites settled on a silicon chip (C, D) or macrophages grown on an aclar coverslip (E, F) prior to mounting on double-sided carbon tape, and stabilized further with Leitsilber silver paint. Scale bars (B & D) = 1 μm and E = 10 μm.

Figure 15

Chlamydia infected HeLa cells were prepared by OTOTO and left uncoated for examination (A, B), minimally coated with 20 angstroms of Cr, (C, D) or conventionally processed and coated with 75 angstroms of Ir (E, F). Scale bars = 0.5 μm.

Figure 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 17

Bacteria in ovarian tissue labeled with Nanogold and silver enhanced for 15 minutes to improve visualization by SEM. Scale bar = 0.5 μm.

Figure 18

Q-dot labeling and detection by SEM. HeLa cells were infected with Chlamydia trachomatis elementary bodies, probed with anti-chlamydial rabbit serum, and labeled with anti-rabbit Q-dots (565nm peak emission). Panels A–C demonstrates examination of clustered elementary bodies on the surface of infected HeLa cells by fluorescence/light microscopy, TEM, and SEM, respectively. Scale bars = 500 nm unless designated).

Figure 19

Scotch tape is gently applied to cell layer grown on Thermanox™ coverslips.

Figure 20

Macrophages infected with Francisella tularensis fractured with adhesive tape to expose internalized bacteria (A), <i>Yersinia pestis</i> attached to internal spines of a flea proventriculus exposed by disruption with syringe tip (B), and malarial parasites invading the apical surface of a mosquito midgut after exposure using a micro dissection scissor (C). Scale bar = 1 μm.

Figure 21

Aligned stereo pair.

Figure 22

Images converted to RGB mode.

Figure 23

Red channel removed from left image.

Figure 24

Red image from right image copied on to left image.

Figure 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 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 microns.

Figure 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 bars = 0.5 micron.

Figure 28

Protein globules adhered to an untreated silicon chip were either rotary coated (tilted +/− 90 degrees, 360 degree rotation) with 40 angstroms of Ir (A) or shadowed at a 15 degree fixed angle with 20 angstroms of Pt followed by rotary coating +/− 85 degrees with 20 angstroms of C (B). Scale bar = 0.5 micron.

Figure 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 micron.

Figure 30

Immune-labeled Staphylococcus epidermidis prepared by conventional EM techniques demonstrates detachment of structure of interest. Scale bar = 0.25 micron.

Figure 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 micron.

Figure 32

Macrophages grown on aclar coverslips, conventionally prepared and coated with 75 angstroms of Ir were imaged by SEM at 2 kV (A), 5 kV (B) or 10 kV (C). Scale bar = 1 micron.

Figure 33

Amyloid protein fibrils were processed by OTOTO and then left uncoated, (A), or coated with 20 angstroms of Cr and examined at 5 kV (B), 10kV (C), 30 kV (D–E). Also examined at 30 kV were fibrils coated with 15 angstroms of Pt and 20 angstroms of C (F). Scale bars = 0.05 micron.

Figure 34

HIV infected T-lymphocyte imaged with 5 kV at a working distance of 5 mm (A) or 17 mm (B). Scale bar = 1 micron.

Figure 35

Images of a macrophage show astigmatism in the x direction (A), y direction (B), corrected (C), corrected and focused (D). Scale bar = 2 microns.

Figure 36

Mouse dendritic cells (A) and Plasmodium infected red blood cells (B) imaged at 2 kV display minor signs of charging seen as flattening or streaking respectively. The macrophage imaged at 2 kV in (C) shows more extreme charging, rendering the image useless. Scale bars = 0.5 micron.

Figure 37

HeLa cell co-infected with VZV and Streptococcus. Scale bar = 1.5 microns.

Figure 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 micron.