33.14
Preparation of Samples for Electron Microscopy
To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam doe…
Biological samples must be prepared for electron microscopy to protect the sample from damage by the powerful electron beam and the low pressure created by the vacuum.
Sample preparation starts with chemical fixation and complete dehydration. Chemical fixatives such as glutaraldehyde or formaldehyde cross-link the proteins, while osmium tetroxide is often used to fix the membrane lipids.
Next, the sample is dehydrated gradually with increasing concentrations of ethanol or acetone to avoid sample shrinkage.
For TEM, samples are embedded in a hard epoxy resin. An ultramicrotome then slices the sample into ultrathin cross-sections that electrons can pass through.
These sections are stained with heavy metals, such as uranyl acetate. Molecules that bind the metals appear darker and provide contrast to the image.
For SEM, samples are further dried using liquid carbon dioxide in a process called critical point drying. Samples are then sputter-coated with a thin layer of conductive metal, such as gold, preventing thermal damage to the sample and providing better image quality.
View the full transcript and gain access to JoVE Core videos
Q1: Why is chemical fixation necessary before electron microscopy?
Chemical fixation stabilizes biological samples by cross-linking proteins and preserving cellular structures before exposure to the electron beam and vacuum. Fixatives like glutaraldehyde and formaldehyde cross-link proteins, while osmium tetroxide fixes membrane lipids. Rapid fixation is critical because sample properties begin changing immediately after removal from their natural environment, such as altered mitochondrial appearance due to decreasing oxygen levels.
Q2: What is the purpose of dehydration in electron microscopy sample preparation?
Dehydration removes water from samples to prevent damage from the vacuum environment inside the electron microscope. Samples are gradually treated with increasing concentrations of ethanol or acetone to avoid shrinkage and structural distortion. Complete desiccation ensures the sample remains stable under low-pressure conditions during imaging.
Q3: How do TEM and SEM sample preparation methods differ?
Transmission electron microscopy (TEM) embeds samples in hard epoxy resin and slices them into ultrathin sections using an ultramicrotome, allowing electrons to pass through. Scanning electron microscopy (SEM) uses critical point drying with liquid carbon dioxide and applies sputter coating with conductive metals like gold. SEM samples are examined on their surface rather than sectioned, providing three-dimensional topographical information.
Q4: What role do heavy metal stains play in electron microscopy contrast?
Heavy metal stains such as uranyl acetate bind to cellular molecules and appear darker in electron microscopy images, creating contrast that reveals ultrastructural details. The differential binding of metals to different sample components produces the contrast needed to distinguish cellular structures. This staining technique enhances image quality by highlighting molecular composition and organization.
Q5: Why is sputter coating applied to SEM samples?
Sputter coating deposits a thin conductive metal layer, typically gold, onto sample surfaces using argon ions. This coating prevents thermal damage caused by electron bombardment and improves image quality by enhancing electron conductivity. The process makes non-conductive samples conductive, allowing better electron interaction and clearer visualization of surface features.
Q6: What is cryofixation and when is it used?
Cryofixation rapidly freezes samples using plunge freezing for small cells or high-pressure freezing for larger tissues, preventing water crystallization that damages ultrastructure. Plunge freezing immerses samples in liquid ethane cooled by liquid nitrogen, while high-pressure freezing applies approximately 2000 bar pressure to decelerate ice crystal formation. This technique preserves native cellular structures better than chemical fixation alone.
Q7: How does negative staining enable direct imaging of small biological molecules?
Negative staining allows small samples like ribosomes, viruses, and proteins to be imaged directly without sectioning by mixing them with electron-opaque compounds such as ammonium molybdate or uranyl acetate. A metal layer forms on the grid except where the sample lies, creating a negative image with contrast from metal distribution patterns. This technique is particularly useful for studying ultrastructural details of bacteria, viruses, and isolated macromolecules.