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Introduction

The scanning electron microscope employs a focused beam of high-energy electrons to visualize the regions of interest on the surface of solid specimens. The signals from electron-sample interactions unveil the information about the sample such as its morphology, chemical composition, and crystalline structure and material. The SEM can image the areas ranging from approximately 1 cm to 5 microns in width with 20X TO 30,000X magnification and 50 to 100 nm spatial resolution. The SEM is capable of analyzing selected point locations on the sample; this enables the qualitative and quantitative determination of the chemical compositions, crystal orientations, and structural details of the sample.

Principle

The scanning electron microscope uses a beam of highly energized electrons for the imaging. As the pressure of the SEM chamber increases, the primary electron beam is scattered in the sample chamber, resulting in the widening of the beam diameter, and a reduction in the resolution. The scanning electron microscope produces signals of different energies in response to the specimen-electron interaction with atoms at various depths within the sample. These signals include secondary electrons (SE), back-scattered electrons (BSE), characteristic X-rays, absorbed current, and transmitted electrons. The secondary electrons are emitted from the specimen surface. Consequently, the secondary electron imaging yields very high-resolution images of the sample surface, revealing the details even less than 1 nm in size. The back-scattered electrons (BSE) are the beam electrons that are reflected from the sample by elastic scattering. They are released from deeper locations within the specimen; therefore, the resolution of the BSE images is less than that of the SE images. Characteristic X-rays are emitted when the electron beam excites out the inner shell electron from the sample, resulting in the filling of the shell with a higher-energy electron and energy release. This type of SEM is used to identify and measure the abundance and distribution of elements in the sample (Jones, 2012).

Apparatus

The scanning electron microscope consists of an electron column, a scanning system, detectors, a vacuum system, and electronic controls. The electron column is comprised of an electron gun and electromagnetic lenses that operate in a vacuum. The electron gun produces free electrons and accelerates them to energies in the range 1-40 keV. The electromagnetic lenses focus the electron beam on the region of interest on the sample. The focused electron beam is scanned across the surface of the specimen with the help of scanning coils. The specimen emits a signal in response to the interaction with the electrons in the form of electromagnetic radiation. The detector collects these signals, amplifies and displays an image on the computer monitor.

Protocol

Sample preparation

  1. Place the silicon chips or coverslips on the experiment shelf with the help of fine-tipped forceps.
  2. Trim the coverslips with the help of scissors on two sides.
  3. Place one sterile coverslip per well in 24-cell well plate. Label the plate with the sample using a marking pen. Plate the adherent cells under sterile conditions.
  4. Remove the media to fix the cells.
  5. Wash the cells with 0.5 ml physiologically appropriate buffer (e.g., phosphate buffer saline or Hank’s balanced salt solution).
  6. Add ∼5 ml primary fixative by dispersing a slow stream of the liquid. Completely immerse the cells in the fixative.
  7. Fix the specimen at room temperature by completely immersing it in primary fixative for 30 to 60 minutes. The primary fixative contains 5% glutaraldehyde in 0.1 M sodium cacodylate or 0.1 M phosphate buffer.
  8. Wash the cells three times for 2 minutes each with ∼5 ml rinsing buffer.
  9. Post-fix the cells with ∼5 ml secondary fixative (e.g., osmium mixture) for 30 to 60 minutes at room temperature.
  10. Rinse the cells once for 2 minutes with ∼5 ml rinsing buffer, and then twice with ∼0.5 ml distilled water for 2 minutes each.
  11. Dehydrate the specimen with a graded ethanol series as follows:

25% ethanol, 1× 5 min

50% ethanol, 1× 5 min

75% ethanol, 1× 5 min

95% ethanol, 1× 5 min

100% anhydrous ethanol 3 × 10 min.

Drying

  1. Place the critical-point dryer (CPD) specimen holder in a solvent-resistant container and fill it with 100% dry ethanol.
  2. Place the coverslip in the slot of the CPD holder.
  3. Remove the trans-well membrane filter inserts by immersing the filter in 100% ethanol.
  4. After trimming the filter, pick up the filter with the help of fine-tipped forceps and place it in the CPD container.
  5. Insert the specimen holder into the CPD instrument filled with 100% ethanol.
  6. Administer the transition agent such as CO2 when the chamber has reached 10

Coating

  1. Place the microscope-specific aluminum stub in the specimen storage container with the help of stub tweezers.
  2. Apply double-sided conductive tape on the stub and remove the protective layer.
  3. Place the dried specimen on the stub.
  4. Apply a thin layer of conductive paint on the specimen.
  5. Put the dried specimen in the sputter coater.

Immune labeling

  1. Grow the cells on the shiny side of the 24-well plate.
  2. Remove the excessive liquid. In a hood, gently add ∼5 ml of 2% to 4% EM-grade paraformaldehyde fixative in 0.1 M phosphate buffer saline (PBS), pH 6.8 to 7.4, by dispensing a slow stream on the side of the well, and avoid the direct pressure on the sample.
  3. Incubate the specimen in the fixative for 20 to 30 minutes at room temperature, then wash it twice, each time for 5 minutes in 0.5 ml rinsing buffer.
  4. Add 0.5 ml blocking buffer (e.g., 1% to 3% bovine serum albumin BSA/phosphate buffer saline) for 10 minutes at room temperature.
  5. Add primary antibody 1:100 in blocking buffer (e.g., 1% to 3% bovine serum albumin/phosphate buffer saline) for 1 hour at room temperature.
  6. Rinse twice, each time for 5 minutes with 0.5 ml blocking buffer (e.g., 1% to 3% BSA/PBS).
  7. Add secondary antibody (e.g., 1:20 colloidal gold) in the blocking buffer (e.g., 1% to 3% BSA/PBS) for 30 to 60 minutes at room temperature.
  8. Rinse three times, each time for 5 minutes with ∼5 ml PBS.
  9. Mount the slides on the microscope and observe.

Capturing the SEM Image

  1. Click on the ‘Auto Focus’ in the SEM software.
  2. Set the magnification to the minimum zoom level of 50X.
  3. Select the ‘fast scan’ mode.
  4. Adjust the stage manually by setting the exterior knobs to display the region of interest.
  5. Adjust and improve the focus using the fine focusing knob to get a focused image.
  6. To optimize image clarity, increase the magnification to the maximum level, and then focus the image with the help of the fine focus knob.
  7. Take the image by pressing the photo button.

Making Measurements Using the SEM Software

  1. Select “M” tool in the ‘Panels’ dropdown list.
  2. Measure the length, area, and angle directly in the SEM software.
  3. Save the images to the computer.

Applications

Examination of viral infection in cultured cells (Aranyi., Fenters., & Tolkacz., 1970)

The scanning electron microscope (SEM) is a powerful tool to detect morphological changes in BSC-1 cells following the vesicular stomatitis virus (VSV) or herpes simplex virus infection. The morphological changes of the cultured cells were then related to the time and duration of infection, and to the virus used. Ten-fold serial dilutions of virus were made in basal medium Eagle (BME), and inoculated into the cultured cells. It was observed that the cytopathogenic effect of VSV infection was decreased after 24 hours of infection as indicated by the rounding of cells. Whereas, clear nucleus and nucleoli were observed in the uninfected controls. No cytopathogenic effect was seen although a few nuclei were swollen in the case of herpesvirus on BSC-1 cells in 24 hours duration. However, after 72 hours of herpes virus infection, many nuclei were swollen and appeared in complex aggregates, indicating the formation of a polykaryocyte. The study validated that the scanning electron microscopy is a valuable imaging tool to visualize the morphological alterations caused by a viral infection.

Assessment of bacteria in soil (Hagen et al. 1968)

Scanning electron microscopy is a widely used imaging tool to view the bacterial presence in the soil. In the study, SEM was employed to observe Bacillus cereus and Staphylococcus aureus in three different soils used as substrates. Both the organisms were detected in the soils at a concentration of 107 cells per gram of soil; as the minimal concentration of microorganisms required for detection with the SEM was between 107 and 1010 cells per gram of soil. The scanning electron microscopy yielded two morphological types and presented that the soils differ in their individual physicochemical properties. It was also found that the addition of a known concentration of a microorganism provides a reliable method to determine the sensitivity of the instrument. It was concluded that the SEM enables the detection of bacterial species in soil.

Three-dimensional imaging of maxillofacial biomaterials (Pabst., Weg., & Ackermann, 2017)

The maxillofacial biomaterials were characterized and visualized using the scanning electron microscopy. In the study, one dental implant, soft tissue matrix, and one bony substitute were used for three-dimensional imaging. The scanning electron microscopy revealed the specific characteristics of the maxillofacial biomaterials such as the surface of the dental implant, the architecture of the collagen matrix, and the geometry of the bony substitute. The three-dimensional characteristics yielded better information regarding the sample proportions, surface, and spatial intersections within the sample. It was concluded that the 3D-SEM could be used to visualize and observe the size ratios, surface morphology, and roughness precisely.

Evaluation of the infection process of mycoparasite (Santana et al. 2017)

Lecanicillium fungicola is a mycoparasite that causes dry bubble disease in Agaricus bisporus mushrooms and leads to significant economic losses in commercial production. The study was conducted to monitor the infection process of L. fungicola in A. bisporus. The process was studied in the mycelium of L. fungicola (LF.1) and three strains of A. bisporus (ABI 7, ABI 11/14 and ABI 11/21). The scanning electron microscopy was used to evaluate the vegetative growth and basidiocarp infection. It was found that the vegetative mycelium of the Brazilian strains of A. bisporus is not infected by the parasite. It was observed in the images that the pathogen can interlace the hyphae without causing any damage, and this interlace contributes to the presence of L. fungicola during the substrate colonization and primordial formation of A. bisporus. Within 16 hours of the infection, the basidiocarp germ tubes form and the beginning of penetration takes place within the 18 hours. The crystals of calcium oxalate produced by the pathogen were also visible. The scanning electron microscopy enabled the observation of the process of colonization and reproduction within the specialized structures of L. fungicola.

Precautions

  • Wear lab coats and single-layer gloves while handling the radioactive material.
  • It is advised to monitor the instrument for x-ray leakage before and after the experiment.
  • Clean and dry the samples before placing them in the vacuum chamber.
  • The samples should be fixed in an appropriate fixative without damaging their structure and optimal environment.
  • Specimens should be properly fixed on a mounting substrate and appropriately coated to reduce artifacts.
  • Carefully clean and wipe the equipment before and after the use.

Strengths and limitations

  • The scanning electron microscopy has a wide array of applications for the detailed three-dimensional and topographical imaging of the specimens.
  • The SEM is easy to operate with the proper training and advances in computer technology making the operation user-friendly.
  • The scanning electron microscopy works fast and completes the analysis in less than five minutes. Furthermore, technological advances in modern SEMs enabled the generation of data in digital form.
  • The SEMs are expensive, large and should be housed in the lab area free of any electric, magnetic or vibration interference.
  • Its maintenance requires a steady voltage, currents to electromagnetic coils and the circulation of cold water.
  • The SEM is limited to small, solid, inorganic samples that could fit inside the vacuum chamber and could handle moderate vacuum pressure.

References

  1. Pabst., M. WEG., & M. Ackermann. (2017). Three-dimensional scanning electron microscopy of maxillofacial biomaterials. Br J Oral Maxillofac Surg, 55(7), 736-739.
  2. , E. J. Hawrylewicz., B. T. Anderson., V. K. Tolkacz., & M. Cephus. (1968). Use of the Scanning Electron Microscope for Viewing Bacteria in Soil. Appl Microbiol, 16(6), 932-934.
  3. , J. Fenters., & V. Tolkacz. (1970). Examination of Virus-Infected Cultured Cells by Scanning Electron Microscopy. Appl Microbiol, 20(4), 633-637.
  4. Fischer., B. T. Hansen., V. Nair., F. H. Hoyt., & D. W. Dorward. (2012). Scanning electron microscopy. Curr Protoc Microbiol, Chapter 2. Unit 2B.
  5. C. Jones. (2012). Scanning electron microscopy: preparation and imaging for SEM. Methods Mol Biol, 915, 1-20.
  6. Santana., R. Brito., D. C. Zied., A. Graças Leite., S. E. Dias., & E. Alves. (2017). Evaluation of the infection process by Lecanicillium fungicola in Agaricus bisporus by scanning electron microscopy. Rev Iberoam Micol, 34(1), 36-42.

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