W9 Basics of Electron Microscopy Flashcards
definition of resolution
the smallest distance between two points that can be differentiated
human eye has resolution of about 0.1-0.2mm
basic component of electron microscope
electron gun: produce electron beam
accelerator stack: provide strong voltage to equip electrons with very high speed
condenser lens system: to direct and focus the electron beam onto the sample
sample chamber
objective lens: produces a magnified real image of the sample and is used for focussing
projector lens: allow easier control of the magnification and projects magnified image onto a detector
fluorescent screen
difference between TEM and SEM
TEM:
- detect electrons that are transmitted through sample
- internal structure observation
- passes through the sample
- 2D, high resolution
- thin samples
SEM:
- detect secondary electrons, backscattered
- surface structure observation
- scans the surface
- 3D like, detailed surface view
- bulk samples
what is vitrified water
water that has been transformed into a glassy, solid state without forming any ice crystals
if crystals formed > forces sample molecules to be reorientated and distort its native conformation
sample usually vitrified in liquid ethane
properties of x-ray crystallography
protein to study must be turned into crystal first (must be very concentrated, 10-20 mg/ml, to ensure got enough molecules to arrange into repeating pattern)
shine x-ray onto crystal > x-ray interacts with electrons in the atoms of protein and scatter in specific patterns
diffraction pattern captured using detector > create complex image of dots > analyse diffraction patterns and build 3D model of protein structure
what is the rayleigh criterion
rayleigh criterion for the diffraction limit to resolution states that two images are just resolvable when the center of the diffraction pattern of one is directly over the first minimum of the diffraction pattern of the other
difference between electron and photon
electron has shorter wavelength than photon
advantage and disadvantage of visible light
A: no damage to sample, easy to focus and can be detected by eye
D: long wavelength (~400nm)
advantage and disadvantage of X-ray
A: small wavelength (0.01-10nm) and have good penetration
D: difficult to focus and can damage sample
advantage and disadvantage of electron
A: small wavelength (pm) and can be focused
D: poor penetration and can damage sample
advantage and disadvantage of neutron
A: good penetrating power and small wavelength (pm)
D: difficult to produce and focus well
advantages and disadvantages of electron microscopy
A: much higher magnification (x50000) and greater resolving power (0.1nm)
D: costly, size of instrument, complicated operation, image acquisition needs to be done in vacuum
difference between elastic and non elastic scattering
elastic: scattered electrons can change their direction but do not change their wavelength since no loss or gain in energy
inelastic: occurs when there is loss of energy > increase in wavelength
what can happen to electrons when electron beam hits a sample
- elastic scattering: deflected without losing energy > used for imaging in TEM and STEM
- inelastic scattering: when electron transfers some of its energy to sample > leads to generation of secondary electrons > used in SEM and X-ray
- unscattered electrons: electrons pass through sample without any interaction
- backscattered electrons: high energy electrons that bounce back from sample
- specimen current: the flow of electrons collected from sample due to inelastic scattering
limit of x-ray crystallography
many proteins especially transmembrane proteins very hard to crystallise
proteins with conformation heterogeneity (different shapes) hard to crystallise as molecules need to have same shape to form proper crystal
properties of NMR
studies molecules in their natural state, dissolved in liquid
sample must have concentration of 0.1-1 millimolar
NMR looks at magnetic properties of specific atomic nuclei (usually those with odd number protons or neutrons)
works best for small proteins (<50kDa)
Advantages: can observe how proteins move or change shape overtime and can study how proteins bind to other molecules
properties of Cryo-EM
uses cryogenic sample: sample is frozen at very low temp to preserve natural structure
concentration of 1mg/ml and uses 3microlitre for imaging
can directly capture image (unlike other two that rely on diffraction patterns or magnetic signals)
ideal for large proteins (>200kDa)
can use for large protein complexes, membrane proteins and proteins with conformation heterogeneity
different levels of radiation damage
primary: formation of secondary electrons and free radicals and broken chemical bonds in a few molecules
secondary: formation of secondary electrons, free radicals, and broken chemical bonds in all molecules
tertiary: bubbling of specimen, formation of free radicals and hydrogen gas
what is single particle analysis
computation technique used in Cryo-EM to determine 3D structure of molecules by analysing multiple 2D images of individual particles
millions of particles imaged using EM > each 2D projection shows view of particle from specific angle > aligned to group similar ones together > construction to make 3D structure
what is tomography
to study 3D structures of larger and more complex samples in their native environment
sample is tilted at different angles other than collect series of 2D projections > reconstructed into 3D model
difference between single particle analysis (SPA) and tomography
SPA: works with isolated particles and focuses on obtaining high resolution 3D structures from single molecules
tomography: used for more complex samples and involve creating 3D models from many 2D images taken at different angles
different types of electron guns
heated tungsten: tungsten heated to high temp > e gain enough energy to escape from metal surface > free e accelerated by electric field into a beam (low brightness and less control over e beam)
heated lanthanum hexaboride (LaB6): heated > LaB6 material releases e > accelerated into a beam (higher brightness and sharper e beam but more expensive)
tungsten field emission gun (FEG): strong electric field pulls e from tungsten top without need for heating > e tunnel out of surface into a beam (much higher brightness and sharper e beam but requires high vacuum)
how do magnetic lens work to control e beam
electric current passes through coiled wire > creates magnetic field around wire
magnetic field from coil guides e beam > e moves through magnetic field in a circular motion
adjust current in wire > can control strength of magnetic field > allow e beam to be focused or deflected
purpose of a vacuum in EM
molecules in air can collide with e > e scattering > blurs image > use vacuum to remove air molecules
vacuum also helps to achieve spatial and temporal coherence
different types of detector
fluorescence screen: e hits fluorescence screen > screen emits light > captured using camera
photographic film: e interacts with film > expose film and leave image that can be developed
charged coupled devices (CCD): converts electrons into photons > captured by CCD and converted into electronic image
direct electron detector: directly count e as charge > charge recorded
why is data in cryo-EM recorded as a movie
beam induced motion: e beam interacts with sample > movement of vitrified ice > blurry image
sample stage drift: sample stage may drift > blurry or misaligned images
to solve above 2 issues > multiples frames captured overtime instead of a single still image > frames aligned to compensate movement of sample
what is contrast transfer function (CTF)
mathematically describes how aberrations in TEM modify image of sample
defocus: when lens not focussed properly and spherical aberration: when lens causes e beam to spread out a little
these imperfections make image fuzzy or misleading > correction during data processing to undo the blurriness
