Lecture 8 Cryo-EM

Question 1

Single particle analysis Advantages

Answer 1

• Does not require crystals
• Samples can be partially inhomogeneous
• Physiological conditions possible
• Requires small amount of sample
• Rapid - many steps automated
• May eventually even be possible in vivo

Question 2

Single particle analysis limitations

Answer 2

• Radiation damage
•Precision of image alignment
• Numbers of particles averaged
• Conformational heterogeneity
• Orientational preferences

Question 3

Transmission electron microscopy (TEM)

Answer 3

• Records differing energies electrons possess after they pass through a specimen stained with heavy metal.
• Can visualize small detailed structures like proteins and component of viral particles.

Question 4

Scanning Electron Microscopy (SEM)

Answer 4

• captures the secondary electrons emitted from surface of samples thatare coated in gold or palladium ions
• Allows for visualization of the topography of the specimen

Question 5

Types of radiation

Answer 5

Visible light
X-rays
Electrons
Neutrons

Question 6

Visible light advantages & disadvantages

Answer 6

A:
Not very damaging
Easily focused
Eye wonderful detector
D:
Long wavelength

Question 7

X-rays advantages & disadvantages

Answer 7

A:
Small wavelenghts
Good penetration
D:
Hard to focus
Damages samples

Question 8

Electrons advantages & disadvantages

Answer 8

A:
Small wavelenghts
Good penetration
D:
Damage sample
Poor penetration

Question 9

Neutrons advantages & disadvantages

Answer 9

A:
low sample damage
small wavelenghts
D:
Hard to produce in controlled ways
Hard to focus

Question 10

Resolution

Answer 10

The act, process, or capability of distinguishing between two separate but adjacent objects or sources of light, or between two nearly equal wavelenghts.
Resolution is not increasing with the magnification, it can only increase with the change of numerical aperture.

Question 11

Resolving power

Answer 11

The ability to make points or lines which are closely adjacent in an object distinguishable image.

Question 12

Depth of field

Answer 12

A measure of how much the object that we are looking at remains in focus at the same time

Question 13

Depth of focus

Answer 13

Refers to the distance over which the image can move relative to the object and still remain in focus.

Question 14

Light vs EM

Answer 14

Light:
Illumination source: Ambient light
Lens type: glass
Magnification: moving the lens
Sample observation: ocular
condition: ambient
EM:
Illumination source: Flux of electrons
Lens type: EM
Magnification: Focal lenght is changed by current
Sample observation: Fluorescent screen camera
condition: vacuum

Question 15

Elestically scattered electrons

Answer 15

The elastic scattering occurs with no change in photon energy
if the light scattered has same wavelength and frequency, as the incident photon, the process is elastic in nature

Question 16

Inelestically scattered electrons

Answer 16

the inelastic scattering occurs with change in photon energy
A small fraction of photons scatter inelastically due to a shift in frequency and hence energy, from the frequency of incident photons by the amount of vibrational energy that is lost or gained by the molecule, known as Raman scattering