W9 Basics of Electron Microscopy

Question 1

definition of resolution

Answer 1

the smallest distance between two points that can be differentiated

human eye has resolution of about 0.1-0.2mm

Question 2

basic component of electron microscope

Answer 2

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

Question 3

difference between TEM and SEM

Answer 3

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

Question 4

what is vitrified water

Answer 4

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

Question 5

properties of x-ray crystallography

Answer 5

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

Question 6

what is the rayleigh criterion

Answer 6

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

Question 7

difference between electron and photon

Answer 7

electron has shorter wavelength than photon

Question 8

advantage and disadvantage of visible light

Answer 8

A: no damage to sample, easy to focus and can be detected by eye

D: long wavelength (~400nm)

Question 9

advantage and disadvantage of X-ray

Answer 9

A: small wavelength (0.01-10nm) and have good penetration

D: difficult to focus and can damage sample

Question 10

advantage and disadvantage of electron

Answer 10

A: small wavelength (pm) and can be focused

D: poor penetration and can damage sample

Question 11

advantage and disadvantage of neutron

Answer 11

A: good penetrating power and small wavelength (pm)

D: difficult to produce and focus well

Question 12

advantages and disadvantages of electron microscopy

Answer 12

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

Question 13

difference between elastic and non elastic scattering

Answer 13

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

Question 14

what can happen to electrons when electron beam hits a sample

Answer 14

1. elastic scattering: deflected without losing energy > used for imaging in TEM and STEM
2. inelastic scattering: when electron transfers some of its energy to sample > leads to generation of secondary electrons > used in SEM and X-ray
3. unscattered electrons: electrons pass through sample without any interaction
4. backscattered electrons: high energy electrons that bounce back from sample
5. specimen current: the flow of electrons collected from sample due to inelastic scattering

Question 15

limit of x-ray crystallography

Answer 15

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

Question 16

properties of NMR

Answer 16

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

Question 17

properties of Cryo-EM

Answer 17

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

Question 18

different levels of radiation damage

Answer 18

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

Question 19

what is single particle analysis

Answer 19

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

Question 20

what is tomography

Answer 20

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

Question 21

difference between single particle analysis (SPA) and tomography

Answer 21

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

Question 22

different types of electron guns

Answer 22

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)

Question 23

how do magnetic lens work to control e beam

Answer 23

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

Question 24

purpose of a vacuum in EM

Answer 24

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

Question 25

different types of detector

Answer 25

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

Question 26

why is data in cryo-EM recorded as a movie

Answer 26

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

Question 27

what is contrast transfer function (CTF)

Answer 27

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