Electron Microscopy IC L11-12 Flashcards
What is the wavelength for an electron accelerated through 100kV?
λ = 0.037 Å
electron wavelength is smaller than light wavelength and the resolution is smaller
Advantages of electron microscopy
λ = 0.037 Å for an electron accelerated through 100kV. Therefore wavelength does not limit resolution (BUT other optical problems do).
Electrons carry charge which enables them to be FOCUSSED using magnetic lenses.
Electron scattering by smaller elements (e.g. C, N, O) is about 10,000X greater than for x-rays.
High electron fluxes can be achieved allowing for rapid data collection.
The sample does not have to be crystalline.
Do the samples have to be crystalline?
NO
Disadvantages of electron microscopy
It is necessary to analyse samples in a vacuum because airborne contaminants will also scatter electrons.
High resolution electron microscopy demands expensive equipment and is computationally time consuming (very demanding).
High energy electron collisions gives rise to sample damage (can’t look at anything living.) Therefore, for protein samples low electron dosage conditions must be used.
Low dosage decreases the signal to noise ratio-cant see anything equivalent to taking a picture in the dark.
Metal shadowing
Fire heavy metals from certain angles shows good constrast
Sample preparation and imaging step 1?
1) Grid Preparation for EM
do not need lots of material - 2µl of sample
use forcets to place a carbon coated copper grid (1atom thick of carbon)
Hydrophobic surface
Glow discharging to make the copper coat more hydrophilic so it associates with the water sample better
Add salt stain since O,N,C,H are not normally very electron dense
Sample preparation and imaging step 2?
2) Rapid freezing in liquid ethane!
Pump gaseous ethane into vessel which condenses into liquid ethane
Instantaneous freezing forms non crystallised ice (vitreous ice) since ice crystals look black on EM
Rapid freezing if you don’t use staining
Sample preparation and imaging step 3?
3) Insert specimen into microscope
negative staining requires blotting paper-sample is blotted and air dried
What are the three flavours of cryo-EM?
Note: in all cases they are frozen (preservation/fixation)
Radiation damage decreases at lower temps
Helps with signal:noise ratio
Less radiation damage means you can put more electrons in and get more contrast
- Single particle EM: Large assemblies, viruses 3.5 - 15 Å resolution
- Electron crystallography: mainly small MEMBRANE proteins 2 - 8 Å resolution
- Electron cryo-tomography: whole cells, organelles, sections 30 - 100 Å resolution
Advantages of single particle EM
Small amount of protein needed - 2µl
No crystallisation necessary
Heterogeneity can be analysed (i.e doesn’t matter if your sample and material isn’t pure and only 90% pure.
You can selectively specify what you want by telling the computer)
Disadvantages of single particle EM
Take many projections from diff positions around the specimen and then combine all 2D images in computer to generate a 3D image
Population of images in all diff orientations
Radiation damage > low signal to noise ratio
Limited resolution
One image per particle
Determining heterogeneity (difficulty for distinction if unknown components are in the sample)
Finding relative orientations of particle images (round object looks same in all directions so difficult to recognise distinct features)
2D electron crystallography
- Often easier to produce compared to 3D-crystals • Near-physiological conditions
- Native-like membrane environment
- Stable for months (or years)
- Small amount of material required (especially compared to 3D crystallography)
- Structure determination to ~7Å comparatively quick
- in situ conformational changes
Name the newest microscope available:
and the new camera?
Titan Krios - latest electron cryo-microscope for high resolution data collection and structure determination $5mill
K2 Summit-new Direct Detection camera for EM and is already making impact on cyro 3D EM $500,000
Are ATP Synthases dimeric?
YES shown using single particle analysis
