TB3 - Cryo-EM Flashcards
Overview of Cryo-EM
Cryo-EM involves samples being placed on a copper grid with a carbon film on top, which is then frozen using liquid ethane. This quick-freezing process prevents water crystals from forming and traps the sample in a layer of vitreous ice, keeping the sample hydrated in a near native state. In this state, biological samples can withstand high vacuums and have improved radiation sensitivity.
What is detective quantum efficiency (DQE)?
DQE is a measure of the combined effects of the signal and noise performance of an imaging system. The higher the value, the more completely you’re obtaining the data.
Compare elastic and inelastic scattering
Of the interacting electrons, some are scattered without energy loss (elastic scattering), but others transfer energy to the specimen (inelastic scattering).
Elastically scattered electrons are bent by a nucleus and experience no change in energy of speed, but a change in direction. They are used to generate contrast. Inelastically scattered electrons experience a change in energy and so a change in velocity
Why are apertures used in cryo-EM?
To remove elastic scattering
What is amplitude contrast?
In amplitude contrast, elastically scattered electrons are lost or removed by aperture, such that regions with high density appear darker. Lack of scattering indicates a less-dense area of the sample, whereas if the object is dense, it scatters a lot more electrons which results in less electrons hitting the detector. This creates contrast.
What is phase contrast?
Phase contrast is a result of interference between phase-shifted scattered waves. This interference alters the probability of each electron being detected at any particular spot on the image plane. Phase contrast is mostly used to achieve structures at atomic resolution.
What is spherical aberration?
involves peripheral electrons and are features of the microscopic lens that causes spreading out of the focal point. It is the blurriness at the edge of an image that is caused by the spherical nature of the lens.
What is chromatic aberration?
caused by the lens focusing rays with longer wavelengths more strongly so that part of the image is formed in a plane closer to the object, resulting in ‘colored’ halos around the edges in the images. To avoid it, a combination of several lens are used.
What is astigmatic aberration?
produced by deviation from axial symmetry in the lens, so that the lens is slightly stronger in one direction than in the perpendicular direction.
CCD vs Direct Electron Detectors
Charged coupled devices (CCDs) are generally not so good and used only in live imaging due to their limited number of colors and thus limited quality. Information is digitized in grey scales from the start.
Direct electron detectors capture the data as movies, meaning you can correct for movements in the molecule via post-processing techniques. This improves the ‘crispness’ of data, hence it’s DQE of 0.8-1. As a result of having improved SNR, less intense electron beams are required and so beam damage is reduced.
What is defocussing?
Defocus is a primitive form of phase-contrast imaging where the microscope’s objective lens is intentionally focused beyond the specimen by a distance of a few microns. It introduces an additional phase shift in the data to the phase shift deriving from the initial scattering of electrons, enhancing phase contrast.
What is the contrast transfer function?
a measure of defocus that is governed by the spherical aberration coefficient (Cs), wavelength (λ), spatial frequency and the defocus value.
Describe the process of sample prep for cryo-EM.
A small volume of a sample is placed on a 3mm EM grid – e.g., copper – and the grid will often have a layer of carbon with holes laid over the grid squares, to support the sample. When plunged into liquid ethane that’s cooled in a bath of liquid nitrogen, the heat capacity of ethane means that the sample is cooled faster than crystals can form. Thus, it is vitrified.
What is dosage compensation?
Where samples have been damaged (often a result of beam damage), you reduce their contribution to the overall average. By correcting for beam damage and movement, you can make images clearer. This varies dramatically between detectors, as we want to maximize data with a high DQE.
What are phase plates?
Phase plates add an additional phase shift, shifting their phase by 90˚, so you get better contrast. This is important for smaller molecules to enhance the contrast as it can be harder to resolve them/identify where particles are.
