Sample Preparation for the

Transmission Electron Microscope

 

 

Electrons travel best in a vacuum and will not penetrate a thick sample. Furthermore, vacuums and liquid water or water vapor are not compatible. Since most biological samples are thick, require air, and are composed mostly of water, it is not possible to observe samples as easily as with a light microscope. Preparation of a sample, for example a tissue biopsy, requires several steps taking several days.

1. First a small sample is collected, no larger than 1 mm cubed. Larger samples must be cut into smaller pieces.

2. The sample is preserved as rapidly as possible in potent chemicals that link the varies components of the cell together and keep the internal structure of the cell intact

3. The sample needs to be sliced into very thin slices. Since most tissue (for example, think of how soft and pliable a piece of liver is) is very soft, it must first be embedded in something stiffer to hold it while it is being sliced. The sample is embedded in liquid plastic, then baked in the oven till the plastic hardens.

4. The piece of tissue, now encased in hard plastic, is ready to be sliced. Each slice must be about 100 nanometers thick. How thin is 100 nanometers, you ask? There are 10,000 slices 100 nanometers thick in a millimeter. To cut a slice this thin requires a special machine, called an ultramicrotome, which is pictured above. It is really nothing more than a fancy baloney slicer. Each time the arm with the sample attached moves past the knife edge, a slice is cut off and it floats free on a puddle of water surrounding the knife edge. The knife edge must be very sharp--sharper than any steel blade can be sharpened. We use freshly broken glass--we all know how sharp that is! We glue a trough, which holds the water, onto the knife, place the knife in the machine and proceed to cut our slices.

5. The electron beam won't pass through much of anything, including glass. Because of this, the slices in the trough are picked up on 3 mm screens rather than glass slides. We can view cells only in the open areas of the screen. Naturally, Murphy's Law insures that the most interesting cells are found in that part of the slice that is resting on a grid bar.

6. The slices are stained with stains that will block electrons. These are usually heavy metals, such as Lead. Densely stained parts of cell appear dark, less stained areas will appear lighter. There are no colors, only shades of gray from black (heavy stain) to white (no stain).

7. We are finally ready to put our sliced sample into the vacuum of the microscope for viewing. We observe our images on a phosphor screen until we see something we want to record. We record the image on a high resolution photographic negative, which we subsequently develop and print in our darkroom.