5. Experimental techniques

Question 1

Flurescence =

Answer 1

a type of luminescence where teh light emitted has a longer wavelength than that abosrbed

Question 2

Stokes shift =

Answer 2

the differnce in energy between the emission and absorbtion

Question 3

Kasha’s rule =

Answer 3

the emission spectrum is independent of the excitation wavelenght

Question 4

quantum yield =

Answer 4

the fraction of emitted to absorbed photons

Question 5

fluorescence lifetime =

Answer 5

the overall observed lifetime

In the absence of FRET: t_obs = 1/ (k_r + k_nr)

Question 6

What scale can FRET measure?

Answer 6

2-10 nm

Question 7

How does FRET work?

Answer 7

An excited donor fluorophore can transfer energy to an acceptro through non-radiative dipole-dipole coupling

Question 8

FRET efficiency =

Answer 8

1/(1+(R/R_0)^6)

Where R_0 is the Forster radius

Question 9

How is FRET efficiency measured?

Answer 9

By analysing the joint flurorescence spectra of the donno-acceptor pair. The greater the efficiency, the higher the high wavelength peak.

Question 10

Benefit of a single molecule approach =

Answer 10

no averaging is required - can detect static and dynamic heterogeneity

Question 11

static heterogeneity =

Answer 11

where an ensemble contains distinct, non-interconverting species ie active or inactive states, different conformations

Question 12

dynamic heterogeneity =

Answer 12

where a single molecule can interconvert into differnt states eg active or inactive

Question 13

To maximise fluorescence signal (7)

Answer 13

1. Optimise the fluorophore
   
   1. Bright
   2. Photostale
   3. Large stokes shift
   4. Short excitation lifetime
   5. Minimal triplet state bottlenecks
2. Optimise microscope
   
   1. Maximise collection efficiency
   2. Use detectors with high quantum efficiency

Question 14

To minimse fluorescence experiment noise (6)

Answer 14

1. Confine the excitation
   
   1. tightly focus the laser beam
   2. Evanescent wave (excitation close to surface)
2. Reduce background
   
   1. Spatial filtering - use confcal optics
   2. Spectral filtering - reject scattering
   3. Reduce impurities
   4. Low dark current in the detector

Question 15

How does point detection work?

Answer 15

Use confocal optics to only excite a very small region. Then scan over whole area of interest.

Question 16

What features can we observe wiht single-molecule fluorescence (5)?

Answer 16

1. High precission molecular localisation
2. Stoichiometric data - can find the number of molecules in a functional domain
3. Orientaion of protein domains
4. Nanoscale distance (FRET)
5. Changes in the above

Question 17

FWHM of a diffraction pattern is

Answer 17

lambda/(2nsin(theta) = lambda / 2NA

Question 18

How can a single molecule be localised from its point spread function?

Answer 18

By fitting to a 2D Gaussian

Question 19

How was myosin V walking model deduced?

Answer 19

Using FIONA - fluorescence imaging with one nanometer accuracy

1. One foot was tagged
2. Tagged myosin added to actin
3. ATP added
4. Step size meaasured

Expected step size for hand-over-hand double the expected step size for inchworm

Question 20

Name three methods for breaking Rayleigh criterion

Answer 20

STED - STimulated Emission Depletion

PALM = PhotoActivated Localisation Microscopy

STORM = STochastic Optical Reconstruction Microscopy

Question 21

How does STED work?

Answer 21

A doughnut shaped STED beam surrounds the laser beam. The STED beam has a longer wavelength than the excitation beam. This means the STED beam causes stimulated emission, suppressing flurescence around the imaging point

Question 22

How does PALM work?

Answer 22

1. A molecule is switched on by illuminating it at the correct wavelength.
2. The molecule is imaged and localised
3. The molecule is photobleachd - this is a photchemical process that means the flurophore is permanently unable to fluoresce.
4. Process is repeated for more molecules

This prevents an accumulation of active fluorophores in the sample.

Question 23

How does STORM work?

Answer 23

Similar to PALM but instead of being permanenelty bleached, the flurophores can be switched off by a high power laser.

Question 24

How can diffusion be tracked?

Answer 24

Image and localise single molecule. Plotting the mean diffusion distance against time tells you what kind of diffusion is used ie normal diffusion, correlated diffusion etc

Question 25

Derive the angular deflection of a laser beam in an optical trap

Answer 25