3.3: Electron Microscopy Flashcards
(20 cards)
Transmission electron microscopy
only microscopy to independently result in structure model
Lenses: goal, characteristics, image properties, lens eq, magnification
refocus light/e- for images.. converts diverging ray into converging ray
characterised by: Diameter D, focal length
real or imaginary:
> real can be captured, image only real at focus of ALL rays (f)
> imaginary at all other points
lens eq:
1/u + 1/v = 1/f
u - distance of object v - distance of real image
Magnification:
M = v/u
Refractive Index
n = c/v (velocity of light in vacuum/velocity of light in medium)
typically >1
bottom part of ray reaches slow medium first and is slowed down resulting in bending.
amount of bending depends on refractive index/n
Electron Microscopy: Electrons
highly accelerated electrons used.
electrons have wave-particle duality
> particle = scattering
> wave = diffraction, interference, phase contrast
shorter wavelength than X-rays and light
>100kV to 300kV
resolution not limited by wavelength
Electron interactions with matter (fundamental to TEM) and comparison to X-rays
result in:
elastically scattered electrons
transmitted electrons
> both used in Transition EM
Lower radiation damage per useful elastic scattering than X-rays
Probability of scattering inc with..
scattering angle dec
atomic number of material inc
electron energy dec
100kV vs 300kV electrons in terms of scattering and what they are good and bad for
300kV used for thicker samples to reduce to amount of scattering and absorption
good for: thin/individual particles,
bad: thick or crystals
due to shorter path length/wavelength
Scanning Electron Microscopy
highlight focused electron bean scans sample in xy
scattered and transmitted electrons detected
> good for surface visualisation
Transmission Electron Microscope
Electrons accelerated (100kV-300kV)
uses electromagnetic lenses
vacuum conditions
> reduce air scattering
TEM: Electron source
- Thermo-Ionic (100kV)
heated tungsten filament
larger area of emission limits e- quality - Field Emission Gun (FEG) (200kV-300kV)
smaller source, lower e spread
TEM: Electromagnetic Lenses
Magnetic Lens: consists of
pole pieces, surrounded by copper coils
focused up to 1mm-1nm
require: water cooling due to resistive heating
TEM: Detector
Fluorescent Screen, CCD Camera
Direct Electron Detector
> increased readout rate
TEM: Sample requirements and why
also problem with bio samples
Vacuum stable
> e- beam requires vacuum
Very thin: <500nm
> due to e- short path length
Problem with biological samples: Radiation damage
> Cryo-cooling to help mitigate
Samples prepared on electron microscopy grids (made from copper)
> Reduce heat
TEM: Image formation for bio samples
very thin bio samples in aq. solution have very low contrast
> due to e- interacting with water/bio similarly
Phase contrast is obtained by slight underfocussing and lens aberrations
TEM: Single particle analysis (SPA)
what is it and resolution
analysis of large number of randomly orientated particles
averaging ensemble
resolution: typically 3-4 Angstrom
TEM: SPA
Sample preparation
many copies of identical or related particles in random orientation in thin layer of amorphous ice (vitrified)
TEM: SPA workflow and result
- locate individual particle projections BEFORE averaging
- classify in 2D by similarity
- generate 3D model
- assign orientations in 3D
Direct Result:
3D Coulomb Potential Map
> no phase problem no iteration required
TEM: SPA
3D classification
pre-requisite for high resolution
only apply to regular structures
TEM: Cryo Electron Tomography
tilt-series imaging under low dose of singular thin sample
TEM: Cryo Electron Tomography
Segmentation and Resolution
Manual or automated segmentation/ Identification of individual objects
Optional: Subtomogram averaging
> averaging of multiple occurrences of particles
Resolution: 10-30 Angstrom
6-8 Angstrom with averaging
