Electron Microscopy 1 Flashcards
Name the 5 variations of cryoEM.
- Electron cryo-tomography
- Subtomogram averaging
- Single particle analysis
- 2D electron crystallography
- MicroED
Describe scanning electron microscopy.
SEM detects scattered electrons from the surface and this produces images of the sample.
Describe transmission electron microscopy
TEM gives 2D projection images through the 3D sample.
How much sample is required for cryo-EM?
~3ul
Describe sample preparation briefly.
3ul of sample is placed onto an copper grid, with a carbon layer. Blotted filter paper to remove excess sample. It is the plunged into liquid ethane to cool it to liquid nitrogen temperature.
Why does the sample need to be cooled rapidly?
To prevent water crystals forming, producing artefacts on the image.
What is the disadvantage of sample preparation in cryo-EM?
The sample is not protected in any way, so have to limit the electron dosage.
Give a definition of a Fourier transform in a sentence.
The breaking up of a complex waveform into its composite factors (cosine and sine waves).
Give an analogy that describes how the brain uses the Fourier transform.
Different frequencies of sound resonate in different parts of the cochlea in the ear. This stimulates different neurones, effectively the brain is separating out the waves that form the complex wave.
What are the 3 parameters of a wave?
- Amplitude
- Wavelength (frequency - reciprocal)
- Phase shift
How is the amplitude of a wave represented in a pixel?
By intensity of the pixel.
What are the equivalent axis’ in reciprocal space? (x and y)
h and k, respectively
If you filter out high spatial frequency waves, what will be the general appearance of a picture?
A blurry image, with no fine details.
If you only have high spatial frequency waves, what will be the general appearance of a picture?
A very dark image, with very fine details.
If you take two pictures (picture 1 & 2) and combine the phase of picture 1, with the amplitude of picture 2, which picture will the final image look like?
(what does this tell you?)
Picture 1 - phase is the most important factor for defining the image.
