6: SEM Imaging of Biological Samples

1. Sample Preparation

1. Wear gloves and take precautions to avoid contamination when handling the sample.
2. Make sure that the sample on the slide is dried and there is no contamination on the sample. This is because SEM measures surface characterization, and these defects can severely hinder the signal.
3. If the sample is loaded on a standard glass slide, decrease the size of the sample by scoring the slide with a diamond tipped glass cutter in a straight line and gently push on the scored line away from the body until the glass fractures.
4. Depending on the sample, choose a coating that does not have the same elemental composition (it would hinder the signal received by EDS). For this demonstration, a gold-palladium coating is used.
5. Use the sputter coater as directed. Let the machine sputter the sample for around 40 s for a thin coating with adequate coverage.
6. Mount the sample onto an SEM stub using conductive double-sided carbon tape. This tape should also be placed from the stage to the top of the slide that was sputtered to ground the sample if it is mounted on a non-conductive slide. A thin layer of conductive silver paint could also be used to increase the conductivity of the sample to the stage.
7. Mount the stub onto the stage and tighten the screw on the side.

2. Imaging Procedure

1. Load the stage into the chamber. Shut and seal the door. Then hit the "Transfer" button to open the passage from the loading chamber to the vacuum.
2. Once the transfer button stops blinking and the internal door is open, the sample can be moved into the vacuum chamber by screwing in the metal rods and pushing the sample into the chamber.
3. Unscrew the rod, retract and secure the rod fully into the load chamber, and press "store" to close off the load chamber from the vacuum chamber. The sample is now loaded and the rest of the process takes place from the computer workstation.
4. Move the stage using the controller and by opening the stage navigation panel until it is in your field of view.
5. Move the sample vertically until the working distance is 5-10 mm. When moving the stage in the z-direction, turn on the chamber camera to ensure your sample does not get to close to the electron gun.
6. Turn the extra high tension (EHT) beam on. Note that you may also need to open the column if it has been off for a while.
7. Select the SE2 signal from the detector options.
8. Use a kV setting of about 5 kV for initial imaging, and then increase to 20-30 kV for more signal using the back-scatter mode. If the sample wasn't coated, keep the keV low to prevent too many charging artifacts in the image and to prevent sample damage.
9. If there is no clear image, turn the focus, brightness, and contrast knobs until a structure is visible. This will be a reference for refinement.
10. Turn the focus knob on coarse mode until an image is visible. Then switch to fine to find the best focus.
11. Use stage navigation (not in the z-direction) and the magnification to find an area to save an image from.
12. Decrease the scan speed and turn on line averaging to acquire a better image for saving.
13. Save the image by right clicking and saving to a file location.
14. Insert the BSD and move the stage back to a z-position where the sample is focused.
15. Repeat steps 8-11 while looking for areas of contrast that indicate a higher atomic number.
16. Remove the BSD when done.
17. When ready to remove the sample, hit the button "exchange".
18. Move the sample back into the load chamber and hit "store" then "vent".

Scanning electron microscopy, or SEM, is often used to image biological materials on the nano scale. Optical microscopes, which use light to image a sample, are heavily used to non-destructively image biological samples, however, their resolution and depth of field is limited, thus SEM is used in order to achieve higher resolution down to one nanometer.

In SEM, a beam of electrons is focused through a series of condenser lenses, which then hits the sample. When the beam hits the sample, electrons on the surface are scattered and measured by the detector.

In this video, we will discuss how SEM works, demonstrate how to image a biological sample in the laboratory, and finally, introduce some techniques used to image sensitive samples.

A scanning electron microscope uses a high energy electron beam, which is generated by an electron gun fitted with a filament cathode. The generated electrons are propelled toward the anode and then focused using condenser lenses before entering the objective lens. The objective lens is calibrated to focus the beam on the sample, where it is raster scanned across the surface. The interactions of the electrons with the atoms in the sample are used to study the topography, elemental composition, and crystallinity of the sample. When the incident electron beam hits the surface, it emits secondary and backscattered electrons. Secondary electrons are low energy electrons that are emitted from the sample close to the surface and provide topographical information.

Backscattered electrons, on the other hand, are reflected in the opposite direction of the incident beam. The interaction intensity increases with increasing atomic weight, enabling the user to distinguish compositional differences. Special consideration is needed to image biological samples with SEM, since SEM utilizes a high vacuum, thus biological samples, which typically have a high water content, must be dried first. This can cause collapse of the structure of sensitive samples, especially cells. Thus, cells are treated with a fixative, rinsed, and then dehydrated slowly by washing with increasing amounts of ethanol.

For rigid biological materials, such as the collagen-hydroxyapatite tissue scaffold used in this demonstration, the sample is dried over a period of several days under high vacuum.

Finally, since typical SEM imaging requires a conductive surface, biological samples are often sputter coated with a thin layer of metal prior to imaging. Now that we've discussed how SEM works and how to prepare a biological sample for imaging, let's take a look at how to prepare and image a collagen-hydroxyapatite tissue scaffold.

First, mount the biomaterial sample onto an SEM stub using conductive carbon tape and ensure that the sample is dry and has no contamination on the surface.

Then, place the mounted sample in the chamber of a sputter coater, pump down the chamber, and sputter coat the sample for around 40 seconds to achieve a thin, four- to six-nanometer thick coating of metal, in this case gold, with adequate coverage. Once coated, remove the sample and use conductive tape to connect the stub to the top of the sample, which is now coated with conductive metal.

Finally, mount the stub on the SEM stage and tighten the screw on the side. Now the sample is ready to image with SEM. First, load the stage into the SEM chamber and seal the door, then hit the transfer button to open the passage from the loading chamber to the vacuum. Once the internal door is open, screw the metal rod into the stage and push the sample into the vacuum chamber, then unscrew the metal rod and fully retract it into the load chamber, then press store to close the vacuum chamber.

Now let's image the sample using SEM.

First, move the stage using the controller and navigate the sample into the field of view, then move the sample vertically until the working distance is five to 10 millimeters. Turn the electron beam on and select the detector for secondary electrons, set the beam to five kiloelectron volts initially, then increase up to 20 to 30 kiloelectron volts as needed. If the image is not clear, turn the focus, brightness, and contrast knobs until a clear image appears.

Use the stage navigation and the X and Y directions to locate a new spot on the sample, then increase the magnification until the desired features are visible. Adjust the focus, contrast, and brightness as needed to improve the image quality. You may need to decrease the scan speed and turn on line averaging to acquire a better image, then save the image.

The SEM images reveal a highly three-dimensional and porous structure with fibrous features smaller than 25 microns. These features would be difficult to visualize using optical microscopy, as optical microscopy has a much lower depth of field.

There are many challenges associated with the imaging of biological structures with SEM, including structure collapse or damage from the high energy electron beam. Let's now take a look at how the general SEM technique is applied to these types of sensitive samples. Delicate biological structures, such as these young plant tissues, or those with a high water content, must be treated through a fixation process prior to imaging.

These floral meristems were immediately treated with a freshly prepared formalin/acetic acid fixative solution. The fixed tissue was dissected in ethanol, placed in a mesh container, and dehydrated through an ethanol series of 70%, 80%, 90%, and 100% ethanol. Finally, the plant tissues were dried using a critical point dryer, mounted, and sputter coated with a thin metal coating.

After SEM imaging, it is clear that the untreated structures were heavily damaged by the drying process and showed considerable collapse in structure, while those that were fixed maintained their native structure. Alternatively, cells and other high water content specimens can be imaged using environmental SEM, or ESEM. ESEM utilizes a high energy electron beam that is raster scanned over the sample, as with conventional SEM, however, it enables the imaging of wet or uncoated samples by maintaining a gaseous environment within the chamber.

This is done by separating the high vacuum chamber containing the electron gun from the specimen chamber using two apertures. The electron beam does incur significant losses due to scattering by gas molecules, but is typically a high enough energy for imaging. Here, cells were grown on a silicon chip, functionalized with quantum dots, and fixed using a glutaraldehyde fixation protocol. The cells were imaged in water and show the uncollapsed structure of the cell with individual quantum dots visible on the cell surface.

You've just watched JoVE's introduction to visualizing biomaterials using SEM. You should now understand how SEM works, how biological samples are prepared and imaged, as well as some applications of the technique for sensitive structures.

Thanks for watching!