Electron Microscopy 1 Flashcards
Who invented EM and when?
Ernst Ruska in 1933
Note - did not receive the Nobel prize till 50 years later.
List the sizes of cells, bacteria, viruses, proteins and atoms.
Cells - 10^-5m Bacteria - 10^-6m Viruses - 10^-7/10^-8m Proteins - 10^-9m Atoms - 10^-10m
When did the resolution revolution occur?
Around 2 years ago people started to use EM as a way of resolving atomic structures due to technological innovations.
What is the source of the light microscope?
Light
What is the source of the electron microscope?
Electron gun - made up of a cathode filament and an anode.
What is the job of the cathode?
This is the electron source.
What is the job of the anode?
This is the electron accelerator.
What are the lens in EM?
They are electromagnetic coils.
What is the first lens and what is its job?
First lens is the condenser lens and it concentrates the beam of electrons.
What is the second lens and what is its job?
The second lens is the objective lens and it focuses the beam onto certain parts of the specimen.
Why do we require electrons?
If you want to see objects that are smaller than a photon, then it must be imaged with particles smaller than a photon i.e. electrons.
What is the Rayleigh criteria and what does it define?
d = (0.61 x lambda)/(N.A)
It defines the theoretical resolution limit.
Define all the parameters in the Rayleigh criteria.
d = distance between two points (this is your ability to resolve them).
lambda - wavelength of the radiation
N.A. the numerical aperture of the lens.
What is the only thing that limits the resolution if the rest of the imaging system is perfect?
Diffraction
If the wavelength of visible light is 400-700nm and there is a perfect lens what is the resolution limited to?
~200nm
What is the wavelength of electrons at 200kV?
0.0025nm
However, not possible to get this resolution for a structure
Define an electron.
Electrons are negatively charged particles that interact with other particles.
What type of wave is an electron beam?
A plane wave - the frequency is constant and wavefronts are infinite parallel planes.
What happens when an electron encounters an atom?
Electrons interact (are scattered by) biological material much more strongly than X-rays.
What is the ratio of elastically scattered electrons and inelastically scattered electrons by biological molecules?
3 times as many inelastically scattered electrons as scattered electrons.
What does this bad ratio of inelastic and elastic scattering of electrons implicate?
Produces a bad signal to noise ratio.
How many electrons are unscattered?
80%
What happens when electrons interact with the nucleus?
They are back scattered.
What happens when electrons interact with the electron cloud?
They are either elastically or inelastically scattered.
Which electrons contribute to the image and which ones produce noise in the image?
Elastically scattered electrons contribute to image.
Inelastically scattered electrons produce noise.
What is the signal to noise ratio normally like?
Tends to be poor.
What is bad about inelastically scattered electrons, other than creating noise?
They also produce radiation that is damaging to biological molecules.
Why does EM operate under a strict vacuum?
To prevent unwanted electron scattering - like scattering from air.
What are the two types of electron source?
Field emission gun and thermal emission gun
What does the condenser system do?
Controls intensity and convergence of beam.
What does the objective lens do?
Generates contrast and contributes to the contrast transfer function (CTF)
What does the projector system do?
Magnifies the image.
How are electrons emitted from the source?
The cathode filament is heated with energy and then the electrons are pulled off the metal.
What is the difference between the FEG and TEG?
The FEG has a very small tip so electrons coming off will be very coherent.
What does aperture do overall?
Remove highly scattered electrons - electrons you do not want to contribute to your image.
What does the condenser aperture do?
Reduces spherical abnormalities - reducing spot size.
What does the objective aperture do?
Increases contrast, by removing the scattered electrons that reduce contrast.
How do the condenser and object lens magnets control the amount of electrons that are allowed through?
Through varying hole sizes in the lens magnet.
What are the three electromagnetic lenses in EM?
Condenser, objective and projector lenses.
What are the lens made of and how to they interact with the electrons?
They are lots of copper coils going round that generate a magnetic field that bends the electron plane wave as it passes through the centre of the coil.
What happens to the electrons coming from the condenser system?
They are scattered by the sample situated in the object plan of the objective lens.
What does the objective lens do to the electrons scattered in the same direction by the sample?
Focuses these scattered electrons in the back focal plane producing the diffraction pattern.
What does the projector lens do?
Responsible for image formation - magnifies the image.
What is the objective lens also used for?
Defocussing.
What does defocussing do?
Generates contrast in the image.
What is phase contrast a result of and why is it required?
Result of constructive and destructive interference patterns of electron waves.
The structure of a molecule is imprinted in the small variations in phase of the electron waves.
Why in EM do you not want the image to be taken in focus?
Because all the electron waves are destructively interfering with each other - because all the waves of the positive and negative troughs of the waves cancel each other out.
(remember electrons are plane waves - they are in phase and have same frequency).
What does defocussing allow for?
Allows for contrast to be introduced.
Why does contrast need to be introduced?
Because of poor ratio of elastic to inelastic scattering.
How is contrast introduced?
By introducing a shift in the plane wave
Why does defocussing result in a clearer image?
Electrons are further away from each other when less focussed so there is less interference.
What causes the additional phase shifts?
The objective lens
What is the contrast transfer function?
A mathematical expression describing the phase shifts resulting from defocussing and imperfections inherent in the objective lens.
What is the effect of the CTF on the mage?
Causes oscillations of positive and negative contrast at different resolution (frequency) ranges.
What happens at higher resolution to the contrast?
There are more frequent oscillations between positive and negative contrast.
What does this oscillation cause and how is it resolved?
The oscillation between positive and negative contrast causes huge distortion of the image - to resolve this lenses do Fourier Transforms.
How did the images used to be detected?
after magnification by the projector lens - the image was developed on film in a dark room - very laborious
What was first digital version to be created?
Charged coupled devices (CCD).
What was advantageous about using CCDs?
Much faster and gave instant feedback.
What were the disadvantages of CCDs?
They were less sensitive than using film - the electrons reacted with the silver on the film making it more sensitive.
What is the general process of the CCDs?
Electrons are converted to light and then this is converted to a pixel.
What converts the electrons to light?
A scintillator.
What transfers the light image to the image sensor?
Transferred via fibre optic.
What does the image sensor do?
Converts light image to an electronic image.
Why is CCDs limited in resolution?
Because of scattering of electrons in the scintillator and fibre optic.
What is CCDs an example of?
Indirect detection.
What is direct detection and what does it do to the signal?
Directly converts electrons to pixels - produces a more coherent signal.
Why is it better to use direct detection?
Because if you can detect a single electron interaction with the material then you you do not need as a high of an electron dosage.
What does a lower electron dosage result in?
Less radiation damge.
What can these direct detectors do?
Can align all the images to produce the clearest model - more like recording a movie than taking a photograph.
