What is a Fluorophore?
A molecule that emits light of a certain wavelength after having absorbed light of a different wavelength
What is Photobleaching?
Irreversible destruction of fluorophores due to prolonged exposure to excitation light source
What is the Excitation max. and the Emission max. of a fluorophore?
- Excitation max. - the wavelength at which most absorption occurs by a molecule
- Emission max. - the wavelength at which the most emission occurs by a molecule
Explain why the emission max. is usually a lower energy / higher wavelength than the excitation max.
Energy is lost during electron transitions as heat or kinetic energy
Explain the excited lifetime of a fluorophore - about how many seconds does it last?
- Fluorophore is unstable at highest energy configuration
- Eventually adopts the lowest energy excited state
- Length of time fluorophore is in excited states = the excited lifetime
- Excited lifetime = 10^-15 to 10^-9 sec (very short)
Explain the relationship between the excitation wavelength and the emission intensity in a fluorophore
Emission wavelength is proportional to the amplitude of the excitation wavelength
Explain Stokes Shift
- The distance between the excitation and emission wavelengths
- Is a distinct characteristic of each fluorophore
- Emission wavelengths may overlap between fluors
What impact will the Stokes Shift have on selection of a fluorophore for an experiment?
- Selection of fluorophores should ensure a low overlap (large Stokes Shift)
- Large overlaps (small Stokes Shift) will cause a greater background signal
Draw the absorption and emission spectra of Fluorescein?
What is the absorption max. of Fluorescein?
Absorbance max. = 495 nm
What is the emission max. of Fluorescein?
Emission max. = 520 nm
How is fluorescence measured?
(i.e. how does a fluorescent spectrophotometer work?)
- Xenon lamp sends light to an excitation monochromator
- Excitation laser then passes through lens into the sample perpendicular to detection instruments
- Light from sample then passes through another lens into an emission monochromator
- Light is then detected, amplified, and the data analysed
Outline the factors that influence fluorescence emission or detection
- Excitation light raises a “donor” fluor to an excited state and a photon is released
- An “acceptor” fluor in close proximity (3-30 nucleotides) to the “donor”, emitted photon is absorbed
- Acceptor electron will be raised to an excited state and release a photon at the wavelength of the acceptor fluor (fluorescence)
- If acceptor is not fluorescent, absorbed energy is lost
- Fluorescence emission spectrum of donor must overlap absorption spectrum of acceptor
What does the diagram of FRET, contact quenching and collision quenching look like?
Explain the basis of FRET, collision quenching and contact quenching
- FRET - a technique where energy is transferred non-radiatively from an excited donor molecule to a nearby acceptor molecule through dipole-dipole coupling, typically within 1-10 nanometers.
- Collision Quenching - occurs when the transfer of energy from an excited donor molecule to an acceptor is disrupted by interactions with other molecules, typically due to collisions in solution, which lead to a decrease in fluorescence intensity.
- Contact Quenching - the reduction in fluorescence intensity when the excited donor molecule physically contacts a quenching molecule, leading to energy dissipation without emission of light, typically through chemical bonding or electron transfer.
What is the Forster radius R0?
Distance between donor and acceptor fluorophore for 50% FRET efficiency
What does the Forster radius R0 depend on?
- How parallel dipoles of donor and acceptor are
- Distance between molecules
- Spectral overlap
What is J / the overlap integral?
- Overlap integral between emission spectrum of donor and absorption spectrum of acceptor
- Larger J = higher energy transfer
Explain the relationship between FRET and distance
- Efficiency depends on inverse sixth-distance between donor and acceptor
- sensitive tool for measuring nanoscale distances
Explain the relationship between FRET and dipole moment
The efficiency of energy transfer between the donor and acceptor molecules depends significantly on the relative orientation and overlap of their dipole moments, with optimal transfer occurring when these dipoles are parallel.
What are the main uses of FRET in biochemistry?
- Studying Protein-Protein Interactions
- Structure and Conformation of Proteins
- Receptor/Ligand Interactions
- Immunoassays
- Real-time PCR
Explain how FRET can be used to analyse the interaction between SUMO1 and Ubc9
- Cyan Fluorescent Protein (CFP) is bound to SUMO1 and Yellow Fluorescent Protein (YFP) is bound to Ubc9
- Interaction between the two proteins can be measured by applying light at 414 nm causing fluorescence in CFP
- If YFP is not within 10nm of CFP, then 475nm emission
- If YFP is within 10nm of CFP, then 530nm emission
Draw a diagram showing how protein protein interaction can be detected using FRET and proteins tagged with Green Fluorescent Protein (GFP) and blue (BFP)
Explain how FRET can be used to detect AMP-induced conformational change in EPAC - outline key data related to this
A
* cAMP binds to regulatory domain on Epac
* Epac is tagged at the N-terminal end using CFP and YFP
* FRET between the CFP and YFP tags allow detection of conformational change
B
* Emission spectra of CFP-Epac-YFP excited at 430nm
* Red line = restin
* Green line = 3min after Forskolin treatment
* Forskolin used to raise levels of cAMP
