Tissue Preparation for Electron Microscopy

Summary

This video outlines the meticulous process of preparing tissue samples for electron microscopy, which offers significantly higher magnification and resolution than light microscopy. Key steps include fixation to preserve cellular structure, dehydration, embedding in resin, and ultra-thin sectioning. The sections are then stained with heavy metals for contrast and viewed under an electron microscope, revealing cellular details with striking clarity.

Takeaways

• 🔬 The light microscope uses light and lenses to magnify specimens up to 1,000 times, providing a basic view of cell shapes and sizes but limited cellular detail.
• 🌐 The transmission electron microscope (TEM) offers much higher magnification (up to 1 million times) and resolution, using a beam of electrons instead of light to produce images.
• 🏷️ Fixation is a critical first step in preparing tissue samples for electron microscopy, preserving cellular structure close to the time of cell death.
• 🧪 Common fixatives used include glutaraldehyde and paraformaldehyde, which help stabilize cellular organization.
• 🚿 After fixation, tissues are washed and treated with osmium tetroxide, a post-fixative that preserves cell membranes by reacting with lipids.
• 💧 Dehydration is the process of removing water from the tissue samples using increasing concentrations of alcohol, followed by propylene oxide.
• 🧪 Embedding involves infiltrating the dehydrated specimens with epoxy resins, which harden to support the tissue for sectioning.
• 🔪 Sectioning requires the use of glass or diamond knives to cut thin sections from the hardened resin block, which are then stained and viewed under a light microscope.
• 🖌️ Staining with heavy metals like uranium and lead enhances contrast in electron microscopy by creating electron-dense areas that deflect the electron beam.
• 📊 The thickness of sections is crucial for electron microscopy; only sections 70 to 90 nanometers thick are suitable for viewing.
• 🔍 The final preparation step involves mounting the sections onto copper alloy grids for viewing in the electron microscope, which cannot penetrate glass.

Q & A

What is the primary difference between light microscopy and electron microscopy?

Light microscopy uses visible light and glass lenses to magnify the specimen, while electron microscopy uses a high-velocity beam of electrons and electromagnetic lenses, providing much greater magnification and resolving power.

What is the maximum useful magnification achievable with a light microscope?

The maximum useful magnification achievable with a light microscope is 1,000 times.

How does the electron microscope achieve its high resolution?

The electron microscope achieves high resolution by using a beam of electrons instead of light rays, which allows for much greater magnification and detail visibility.

What is the first step in preparing tissue samples for electron microscopy?

The first step in preparing tissue samples for electron microscopy is fixation, which stabilizes the cellular organization.

What are the two most common fixatives used in the fixation process?

The two most common fixatives used in the fixation process are glutaraldehyde and paraformaldehyde.

What is the purpose of using osmium tetroxide as a post-fixative?

Osmium tetroxide is used as a post-fixative to preserve cell membranes, as it combines with lipids to produce black, insoluble compounds.

What is the role of dehydration in the preparation of tissue samples for electron microscopy?

Dehydration removes water from the tissue samples using increasing concentrations of alcohol, preparing the samples for infiltration with epoxy resins.

Why are glass or diamond knives used for sectioning in electron microscopy?

Glass or diamond knives are used for sectioning in electron microscopy because the resin used to embed the tissue is very hard, and these knives can produce thin, precise sections.

What is the purpose of staining in electron microscopy?

Staining in electron microscopy enhances contrast by using heavy metals like uranium and lead, which deflect the electron beam and create electron-dense areas that appear black, improving the visibility of cellular structures.

Why are copper alloy support grids used instead of glass slides for electron microscopy?

Copper alloy support grids are used instead of glass slides because electrons cannot penetrate glass, and the grids provide a suitable surface for the thin sections to be examined.

What is the final step in tissue preparation for electron microscopy?

The final step in tissue preparation for electron microscopy is staining the sections with heavy metals to enhance contrast and then viewing them with the electron microscope.