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Fixation for Electron Microscopy

Electron microscopes use electrons as opposed to light to produce an image of the specimen being observed. This allows for higher resolution images and higher magnification. Slides that are going to be looked at under an electron microscope need to be prepared a bit differently than those headed for a normal light microscope.


Fixation:

The most important thing to remember about fixation for electron microscopy is that you don’t want to do anything that is going to alter the proteins you’re looking at when zoomed in to the high magnifications and resolutions that electron microscopy is designed for. So, you want to make sure that fixation is prompt to avoid degradation of proteins. You also need to use a non-coagulative fixative to avoid seeing clumping of any proteins. Excessive heat can also denature proteins and a non- physiologic pH can cause swelling or shrinking of membranes, so you need to make sure the solution remains isotonic.


Fixatives:

Aldehydes such as glutaraldehyde may be used for electron microscopy because they are good at preserving structure and because of the quick penetration rate, however aldehydes alone don’t preserve lipids, so a secondary fixative of osmium tetroxide is used for preservation of membranes. Though it is good for lipid preservation, as with most fixatives, osmium tetroxide has its own disadvantages, a big one being safety concerns. It should be used under a hood. Osmium tetroxide also hardens as it penetrates, which means you can’t use it on big tissue because its not going to penetrate all the way before it starts hardening, and you also can’t leave tissue in it too long because of the hardening issue. Osmium tetroxide is unique though in its ability to act as both a fixative and a stain. The tissue will turn black as it is fixing.


Zamboni is another fixative used for electron microscopy. Zamboni is made up of picric acid, formaldehyde, and phosphate buffer (which is used to keep the pH neutral). This is a good dual purpose fixative, which means it can be used for regular light microscopy and electron microscopy. You will see osmium tetroxide used as a secondary fixative here often as well.


Buffers:

As previously mentioned, buffers are used in fixation for electron microscopy to keep the pH neutral. Zamboni uses phosphate buffer which is non-toxic and stable at room temperature but can cause nuclear shrinkage and can’t be used with some metal staining. There are also other buffers used, though some of these are dangerous and/or hard to obtain. For example, cacodylate contains arsenic and veronal acetate contains sodium barbital, which is a controlled substance. S-collidine is another one that is commonly used for osmium tetroxide.


You can learn more about electron microscopy in the HTL Prep Course Module, available on elearn.nsh.org.

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