What is the advantage of using electrons compared to normal light microscopy?
electrons have a shorter wavelength and therefore a lower resolution limit
electrons have the highest information/radiation damage ratio
What is vitrification? Why is it necessary?
vitrification: freezing without crystallization (achieved by fast freezing)
necessary because we need vacuum in the microscope because we use electrons, and liquid water is not compatible with that
if we remove the water and replace it with, e.g., a resin, we also remove a lot of cell contents we want to keep
—> we can preserve the sample in close-to-native state!
Name and explain the steps of a typical cryo-ET workflow.
cell culture
vitrification
cryo-LM
goal: find area of interest in your sample
cryo-FIB
goal: thinning of the sample (100-200 nm)
cryo-ET
What is the missing wedge problem and how can you solve it?
When tilting the sample slice, you cannot image it from 180°. Instead, there is a “wedge” of angles you do not see.
To solve the problem, you can rotate the sample by 90° so that you get a “missing pyramid”. If your sample is a cylinder, you can just rotate it perfectly.
You can also solve the problem by subtomogram averaging (aligning pictures of the same thing from different orientations).
What are major challenges in cryo-ET?
vitrification method
plunge-freezing only suitable for very thin samples, sometimes leads to crystallization anyway
(you can add glycerol to improve this method)
high-pressure freezing works for thicker samples, but you end up with a brick of ice you have to further process
waffle method
—> generally, you need expertise and expensive equipment for everything better than plunge-freezing
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