Even though the electron microscope was invented, biologists still couldn’t see many of the structures. They believed that many features observed in electron micrographs were artefacts, features not present in life.
Freeze-fracturing
This is a different technique for examining existent structures in cells:
• Living material is plunged into liquid nitrogen at -196º and pushed against a sharp blade in a precise way.
• The frozen tissue splits along lines of weakness, often in the middle of a membrane.
• The fractured surfaces are ‘etched’ with heavy metal so that the specimen can be examined by the Transmission Electron Microscope.
Freeze fracturing has confirmed the existence of structures in cells and has also revealed new features.
Cell fractionation
This is a technique used to prepare samples of the various cell organelles so that their functions can be studied. Organelles are separated into fractions according to their size (using differential centrifugation) or density (using density gradient centrifugation). For either method, the tissue is first cut into small pieces and then placed in a chilled, isotonic and buffered solution.
• The Tº of the solution is kept low to slow down metabolism and minimize self-digestion of the organelles.
• The salt concentration of the solution is made isotonic so that organelles do not change volume.
• The solution buffered to minimize changes in PH during the process; this prevents enzymes in the organelles from becoming denatured.
Freeze-fracturing
This is a different technique for examining existent structures in cells:
• Living material is plunged into liquid nitrogen at -196º and pushed against a sharp blade in a precise way.
• The frozen tissue splits along lines of weakness, often in the middle of a membrane.
• The fractured surfaces are ‘etched’ with heavy metal so that the specimen can be examined by the Transmission Electron Microscope.
Freeze fracturing has confirmed the existence of structures in cells and has also revealed new features.
Cell fractionation
This is a technique used to prepare samples of the various cell organelles so that their functions can be studied. Organelles are separated into fractions according to their size (using differential centrifugation) or density (using density gradient centrifugation). For either method, the tissue is first cut into small pieces and then placed in a chilled, isotonic and buffered solution.
• The Tº of the solution is kept low to slow down metabolism and minimize self-digestion of the organelles.
• The salt concentration of the solution is made isotonic so that organelles do not change volume.
• The solution buffered to minimize changes in PH during the process; this prevents enzymes in the organelles from becoming denatured.
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