5. Experimental techniques Flashcards
Flurescence =
a type of luminescence where teh light emitted has a longer wavelength than that abosrbed
Stokes shift =
the differnce in energy between the emission and absorbtion
Kasha’s rule =
the emission spectrum is independent of the excitation wavelenght
quantum yield =
the fraction of emitted to absorbed photons
fluorescence lifetime =
the overall observed lifetime
In the absence of FRET: t_obs = 1/ (k_r + k_nr)
What scale can FRET measure?
2-10 nm
How does FRET work?
An excited donor fluorophore can transfer energy to an acceptro through non-radiative dipole-dipole coupling
FRET efficiency =
1/(1+(R/R_0)^6)
Where R_0 is the Forster radius
How is FRET efficiency measured?
By analysing the joint flurorescence spectra of the donno-acceptor pair. The greater the efficiency, the higher the high wavelength peak.
Benefit of a single molecule approach =
no averaging is required - can detect static and dynamic heterogeneity
static heterogeneity =
where an ensemble contains distinct, non-interconverting species ie active or inactive states, different conformations
dynamic heterogeneity =
where a single molecule can interconvert into differnt states eg active or inactive
To maximise fluorescence signal (7)
- Optimise the fluorophore
- Bright
- Photostale
- Large stokes shift
- Short excitation lifetime
- Minimal triplet state bottlenecks
- Optimise microscope
- Maximise collection efficiency
- Use detectors with high quantum efficiency
To minimse fluorescence experiment noise (6)
- Confine the excitation
- tightly focus the laser beam
- Evanescent wave (excitation close to surface)
- Reduce background
- Spatial filtering - use confcal optics
- Spectral filtering - reject scattering
- Reduce impurities
- Low dark current in the detector
How does point detection work?
Use confocal optics to only excite a very small region. Then scan over whole area of interest.
What features can we observe wiht single-molecule fluorescence (5)?
- High precission molecular localisation
- Stoichiometric data - can find the number of molecules in a functional domain
- Orientaion of protein domains
- Nanoscale distance (FRET)
- Changes in the above
FWHM of a diffraction pattern is
lambda/(2nsin(theta) = lambda / 2NA
How can a single molecule be localised from its point spread function?
By fitting to a 2D Gaussian
How was myosin V walking model deduced?
Using FIONA - fluorescence imaging with one nanometer accuracy
- One foot was tagged
- Tagged myosin added to actin
- ATP added
- Step size meaasured
Expected step size for hand-over-hand double the expected step size for inchworm
Name three methods for breaking Rayleigh criterion
STED - STimulated Emission Depletion
PALM = PhotoActivated Localisation Microscopy
STORM = STochastic Optical Reconstruction Microscopy
How does STED work?
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
How does PALM work?
- A molecule is switched on by illuminating it at the correct wavelength.
- The molecule is imaged and localised
- The molecule is photobleachd - this is a photchemical process that means the flurophore is permanently unable to fluoresce.
- Process is repeated for more molecules
This prevents an accumulation of active fluorophores in the sample.
How does STORM work?
Similar to PALM but instead of being permanenelty bleached, the flurophores can be switched off by a high power laser.
How can diffusion be tracked?
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
Derive the angular deflection of a laser beam in an optical trap
