Samples are prepared for electron microscopy through a multi-step process designed to preserve their structure and enhance their visibility. This often involves fixation, dehydration, embedding, sectioning, staining, and making the sample conductive.
Preparation Steps
The specific preparation techniques vary depending on the type of electron microscopy being used (e.g., Transmission Electron Microscopy (TEM) or Scanning Electron Microscopy (SEM)) and the nature of the sample. Here's a general overview:
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Fixation: This step aims to preserve the sample's structure by cross-linking proteins and stabilizing cellular components. Common fixatives include:
- Glutaraldehyde: Primarily used for protein fixation.
- Formaldehyde: Often used in combination with glutaraldehyde.
- This step prevents degradation and autolysis of the sample.
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Dehydration: Because electron microscopes operate under a high vacuum, water must be removed from the sample to prevent it from collapsing or outgassing. This is typically achieved through a series of increasing concentrations of ethanol or acetone.
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Embedding (Primarily for TEM): The dehydrated sample is infiltrated with a resin (e.g., epoxy resin) that will harden and provide support during sectioning. This resin allows for very thin sections to be cut.
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Sectioning (Primarily for TEM): Using an ultramicrotome, the embedded sample is cut into extremely thin sections (typically 50-100 nm thick). These sections are then mounted on copper grids.
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Staining: Electron-dense stains are used to enhance contrast and improve visualization of the sample's features. Common stains include:
- Uranyl acetate: Stains nucleic acids and proteins.
- Lead citrate: Stains cellular membranes and ribosomes.
These heavy metal stains scatter electrons, creating contrast in the image.
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Making the sample conductive (For SEM and sometimes TEM): Because electron beams can cause charging artifacts on non-conductive samples, samples are often coated with a thin layer of conductive material.
- Sputter Coating: This is a common method for coating samples with a thin layer of gold or platinum. The sample is placed in a vacuum chamber, and argon ions are used to bombard a target of the coating material. This causes the atoms of the coating material to be ejected and deposited onto the sample's surface, making it conductive.
- Carbon Coating: Used when X-ray microanalysis is performed as gold or other metals can interfere with the signal.
Key Differences Between TEM and SEM Sample Prep
| Feature | TEM | SEM |
|---|---|---|
| Sectioning | Required, produces very thin sections (50-100 nm) | Not typically required, although samples can be sectioned for specific analyses. |
| Embedding | Required to support thin sections | Not typically required unless sectioning is performed. |
| Sample Thickness | Extremely thin | Can be bulk material or thin films. |
| Information Gained | Internal structure, high resolution | Surface topography, lower resolution compared to TEM. |
Example
Imagine preparing a biological sample like a piece of tissue for SEM. You would:
- Fix the tissue to preserve its structure.
- Dehydrate it to remove water.
- Mount the sample on a stub.
- Coat the sample with a thin layer of gold using a sputter coater to make it conductive.
