EM - Electron Microscope

Overview

Electron Microscopes are some of the most advanced microscopes available to use. Depending on their power outage they can resolve individual molecules and in some cases individual atoms. A light microscope's resolution is limited by diffraction, this becomes a serious issue at wavelengths between 0.4 – 0.7μm. Electron microscopes use a wavelength of less than 10-5μm.

The best Electron microscope for your analysis will depend on what type of analysis you want to be performed. There are two types of electron microscopes, and their main differences are.

Scanning Electron Microscope (SEM)

SEM is a popular technique as it creates images by detecting the reflected and knocked-off electrons from a sample or specimen. This allows for only surface data to be gathered about a sample/specimen.

For information on the surface of your specimen such as roughness and contamination detection, an SEM system would be preferred.

Transmission Electron Microscope (TEM)

TEM creates its images by electrons passing through the sample.TEM machines offer more valuable information on the inner structure of samples/specimens like internal crystal structures, stress state info, and morphology.

For the analysis of internal structures such as crystal structures or structural defects/impurities, a TEM will be suited for that analysis.

Required materials

• Electron microscope

• Vacuum pump

• Electron source

• Specimen holder

• Glutaraldehyde

• Formaldehyde

• Osmium tetroxide

• Ethanol

• Sticky carbon disc for mounting

• Conductive material coating

Simplified Preparation of specimens for SEM

• Primary fixation with aldehydes Glutaraldehyde and Formaldehyde

• Secondary fixation with osmium tetroxide

• Dehydration with Ethanol

• Drying

• Mounting onto carbon disc

• Conductive coating of specimen

Protocol for SEM

1. Isolate and clean the specimen. This may involve cross-sectioning, excising, or otherwise reducing the size of the specimen so that it will fit in the SEM, and removing any contamination that could damage the microscope (oil, water, other environmental contamination).

2. Determine the area of interest for analysis in the SEM, and mark the specimen so that this area can be easily located when the specimen is in the SEM. Here, light (optical) microscopy can be useful as a first-pass analysis.

3. If needed, sputter coat the specimen with a thin layer of metal. This step is needed if the specimen is electrically insulating to prevent charging, which will introduce artifacts in the image.

4. Place the sample into the SEM, which will normally involve:

   • Mounting the specimen on the sample stage.

   • Bringing the specimen chamber of the SEM up to atmospheric pressure.

5. Load the sample holder into the SEM.

6. Pumping the specimen chamber to the required vacuum.

7. Move the sample into the analysis position, and find the areas of interest using low magnification.

8. At higher magnification, optimize the lens settings (magnification, beam energy, beam size, focus).

9. Collect images at a variety of magnifications, using both secondary and backscattered electrons (where available).

10. Refer to the collected images and analyze them according to research parameters.