cryo E.M
in the preceding section, we covered the why's and how's of freezing a sample. This section explains the actual process of imaging a frozen sample. Also covered are other considerations that must be kept in mind when deciding whether to use cryo EM.
Imaging The Sample & Taking Pictures
While taking a picture of a negative stained sample is straighforward, imaging cryo EM samples is more involved. The sample is so delicate that the only time it can be exposed to the electron beam is when the picture is to be taken. Therefore, all image focusing and alignment must be done off to the side, in an area that is not going to be used. For more information on this low dose technique, see below.
Low Dose
An electron beam is a stream of high energy particles that are bombarding the sample. The image that is viewed is a result of the interaction of the sample with this beam. Most of the electrons that form the high resolution image appear due to elastic scattering, where only their trajectory has been changed, but their energy is unaffected. However, a small fraction of the electrons transfer some of their energy to the sample. This energy accumulates and can break apart molecular bonds, destroying the sample after some time. Therefore, for high-resolution imaging, low dose parameters require that the area to be imaged is not exposed until the picture is actually taken. All image calibration and focusing is done beforehand on a nearby area, in the hope that it's properties are similar to the final imaged area. Also, for the final imaging, very low electron doses on the order of 6-10 electrons per Å2 are used. As a comparison, high-resolution electron microsopy of semiconductors routinely uses doses of 100 thousand electrons per Å2.
As a corollary of using a low electron dose during imaging, the signal to noise ratio of the image is very low. For image reconstruction, a proportionally larger number of molecules must therefore be imaged.
Advantages & Disadvantages Over Negative Staining
What are the advantages of cryo EM over negative staining?
The sample is always in solution and never comes into contact with an adhering surface. Therefore, the shape that is observed is the true shape of the hydrated molecule in solution and has not been distorted by attaching itself and flattening against the supporting film.
There is no stain to distort the sample. Stain does not always lay down evenly, which can generate artifacts and false contrasts when reconstructing the structure of a sample. Also, the staining process requires that the sample be blotted dry. During the drying, the sample can be damaged in many ways, such as by flattening and twisting.
When the sample adheres to the carbon grid, it could stick in a preferential orientation. If this happens, then information will be missing from the final image set (a missing cone), and the resolution of the calculated model in that direction will be lacking.
Low dose methods are normally used. Therefore, the electron beam causes less damage to the sample.
Are there reasons, then, to not use cryo EM?
Very low signal to noise ratio. Biological macromolecules are normally made up of carbon, hydrogen, oxygen, and nitrogen. The electron absorption of such molecules is very low. As a result, image contrast is also very low and it is hard to detect features when dealing with just a few images.
Can not do tilt imaging as well. The ice cross section of a tilted frozen sample is too thick to yield good images. Only untilted images are usually taken. Furthermore, charging is more widespread when imaging a tilted frozen sample.
More time consuming to generate samples. However, this is generally not a big problem, especially once a working protocol is designed and good samples are readily available.
If vitreous ice cannot be easily formed, the resulting cubic ice absorbs electrons very easily and the frozen sample is basically worthless.
Other Considerations
One of the reasons for using cryo techniques in electron microscopy is that the specimen is frozen in time. Thermal vibrations are reduced to a minimum, and the corresponding noise is removed from the images. When a specimen is bombarded with electrons, some of these electrons are absorbed into the surrounding medium (vitreous ice in the case of a good cryo sample). There they energize the ice (also known as charging) and induce vibrations, which can move the embedded sample. This movement reduces the resolution that can be achieved and also damages the sample. To avoid this problem, it is useful to capture part of the holey carbon grid within the electron beam. At high enough voltages, carbon can conduct charges over its surface. When the electron beam charges the ice, the carbon can act as a ground sink, bleeding surface electrons away from the delicate sample
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