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Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research
Lesley Graham1, Jan Marc Orenstein
1Department of Pathology, George Washington University Medical Center, Ross 502, 2300 Eye Street, NW, Washington, District of Columbia 20037, USA.
Nature Protocols
|October 20, 2007
Summary
This study details a transmission electron microscopy (TEM) protocol for preparing biological specimens. The method enhances ultrastructural examination for diagnostic pathology and research, accommodating various sample types and fixation methods.
Area of Science:
- Microscopy
- Cell Biology
- Pathology
Background:
- Transmission electron microscopy (TEM) offers high-resolution ultrastructural visualization beyond light microscopy.
- Ultrastructural examination is crucial for diagnostic pathology and biological research, aiding in the study of cells, organelles, and microorganisms.
- Current methods require specific preparation protocols for accurate analysis.
Purpose of the Study:
- To present a comprehensive protocol for preparing specimens for transmission electron microscopy (TEM).
- To outline requirements for tissue and cell preparation, emphasizing rapid fixation.
- To describe methods compatible with formalin-fixed or paraffin-embedded tissues.
Main Methods:
- Specimen preparation involving fixation, embedding, and sectioning (thin and semi-thin).
- Adaptable protocol for clinical and research samples.
- Consideration for analyzing pre-fixed or processed tissues.
Main Results:
- A detailed TEM specimen preparation protocol is provided.
- The protocol addresses specific needs for tissue and cell analysis.
- Methods for preparing complementary semi-thin sections are included.
Conclusions:
- The presented TEM protocol facilitates detailed ultrastructural analysis of diverse biological specimens.
- This method supports diagnostic pathology and fundamental biological research.
- The protocol's flexibility allows for the analysis of various sample types and fixation states.
Related Concept Videos
01:03Fixation and Sectioning
Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added...
The simplest type of preparation is the wet mount, in which the specimen is placed in a drop of liquid on the slide. A liquid specimen can be directly deposited on the slide using a dropper. Solid specimens, such as skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid is simply water, but stains are often added...
01:15Transmission Electron Microscopy
In 1931, physicist Ernst Ruska—building on the idea that magnetic fields can direct an electron beam just as lenses can direct a beam of light in an optical microscope—developed the first prototype of the electron microscope. This development led to the development of the field of electron microscopy. In the transmission electron microscope (TEM), electrons are produced by a hot tungsten element and accelerated by a potential difference in an electron gun, which gives them up to 400 keV in...
01:20Preparation 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 does not destroy them and dried thoroughly (desiccated/dehydrated) so the vacuum does not affect them. Fixation needs to be done as quickly as possible because the sample properties will start changing as soon as it is removed from its natural environment. For example, in a tissue sample, the oxygen levels begin decreasing, causing an altered...