Larger area sample and when surface information is needed

Prof. Jones: ‘Correct, we use SEM when we need surface information and not inner structure! TEM would be ideal to visualize cell membrane components, cell organelles, proteins, cytoskeleton fibers Also we cannot visualize live specimens with SEM or TEM. This is because TEM samples are fixed with heavy metal stains and SEM samples are coated in gold or palladium ions’

You: ‘How do we prepare the slides for an EM? Is the process the same as the ones we followed for the light microscope?’

Prof. Jones: ‘Actually, the process is much longer and more complex. Let’s outline the main parts.’

1. Fixation: First, you must preserve the biological tissue or cells in their current state. This typically involves using chemical fixatives—often glutaraldehyde—followed by post fixation with osmium tetroxide to stabilize cellular structures.

2. Dehydration: Next, you remove water from the sample through a series of graded ethanol (or acetone) solutions. This step prevents distortion or collapse of delicate structures once you place the sample in a vacuum.

3. Embedding: After dehydration, you embed the specimen in a resin (for TEM) or place it on a suitable support (for SEM). For TEM, the resin provides a solid medium in which you can cut ultrathin sections.

4.Sectioning (TEM only): Using an ultramicrotome, you produce very thin slices—usually around 70 nm thick. These sections are transferred onto grids designed for electron microscopy.

5.Staining: Heavy metals (e.g., uranyl acetate, lead citrate) are used to increase contrast and highlight fine details. These stains bind to various cellular components, making it easier to distinguish them under the electron beam. For TEM grid preparation it is important to use inverted tweezers to hold the grid with the carbon side up. Using a pipette, add the sample on the grid, wait for 60 seconds and use a blotting paper to remove the excess solution. Repeat the process with pure water and blot off the excess solution (washing step) and then repeat the step with heavy metal stain and blot off the stain. Store the grid in a grid box.

6. Mounting and Coating (SEM-specific): If you’re preparing a specimen for SEM, you may secure it on an aluminum stub. You often apply a thin conductive metal coating—like gold or palladium—to improve image quality and prevent charging in the electron beam.

7. Imaging: Finally, you place the prepared sample inside the EM’s vacuum chamber and adjust instrument settings (e.g., beam energy, spot size) to capture the desired magnification level and resolution.

Through these steps you preserve and enhance specimen features, ensuring the most accurate and detailed images.