FRET and Other Energy
Transfers
Patrick Bender
Presentation Overview
 Concepts of Fluorescence
 FRAP
 Fluorescence Quenching
 FRET
 Phosphorescence
Fluorescence
Basically the emission of light associated
with electronic transitions
Absorbs one color light and emits another
Uses:
Tracking molecules (i.e. proteins)
Give information about solute environment
Molecular ruler
Etc.
How does it work?
Excited state
1. (Solid Arrow) Excitation from
impinging photon
2. (Dotted Arrow) Internal conversion
3. (Dashed Arrow) Electronic relaxation
and light emission
Note:
• Emitted light has longer wavelength
than impinging
• Internal conversion really fast
(picosecond vs. microsecond)
Ground state
Fluorescence Quantified
(Quantum Yield)
Number of photons fluoresced
Φf =
Number of photons absorbed
FRAP
Fluorescence Recovery After Photobleaching
Used to examine Brownian motion and
2-D interactions in membranes
Examine molecular transport
FRAP procedure
1. Baseline reading of
fluorescing membrane
2. Photobleach to
destroy fluorescence
in a spot
3. Monitor rates of
fluorescence recovery
4. Fluorescence recovery
http://www.me.rochester.edu/courses/ME201/webproj/FRAP.gif
Fluorescence Quenching
Environmental effect
Solvent
Additional solutes
Other moieties
Drastically effects quantum yield as well
as rate of fluorescence
How does it work?
Fluorophore
Fluorophore
Molecular
Oxygen
Molecular
Oxygen
Fluorescent
Not
Fluorescent
Fluorophore
Iodide
High-energy vibration
states
Fluorescent
Radiationless
energy transfer
Examples of quenching
Ethidium Bromide
Interchelated with DNA vs. in solvent
Interchelated with DNA in presence of other
metals
Fluorescence quenching by tryptophan
Locate fluorophore proximity to tryptophan
Quenchers
Single molecule protein folding
Fluorescing molecules quench each other in
folded conformation
Common quenchers:
Water
Molecular Oxygen
Many electron molecules/ions (e.g. Iodide)
FRET
Forster Resonance Energy Transfer
Involves “radiationless” energy transfer
Used as molecular ruler
Use in photosynthesis
FRET
• Excitation of Donor
• Internal conversion of donor
• Excitation transfer of donor
• Fluorescence of acceptor
What we can calculate
 Efficiency of transfer:
D  A
Eff  1 
D
 Distance between fluorophores (r)
r06
Eff  6 6
r0  r
r0= Distance where efficiency equal 0.5
http://www.olympusfluoview.com/applications/fretintro.html
Photosystem II
Phosphorescence
Emission of light resulting from quantummechanically forbidden transitions
“Glow in the dark”
How it works
S1
Intersystem crossing
T1
S0
Consequences
Violates quantum mechanics selection
rules
Inversion of spin
Lifetime of excited triplet state in the
millisecond or longer range
Uses
Can be used to test for presence of
oxygen species in different environments
Non-invasive
Examine mitochondrial function and energy
levels of cells
Dmitriev, R., Zhdanov, A., Ponomarev, G., Yashunski, D., & Papkovsky, D. (2010). Intracellular oxygen-sensitive
phosphorescent probes based on cell-penetrating peptides. Analytical Biochemistry, 398(1), 24-33.
doi:10.1016/j.ab.2009.10.048.
List of Works Cited
 Dmitriev, R., Zhdanov, A., Ponomarev, G., Yashunski, D., & Papkovsky, D.
(2010). Intracellular oxygen-sensitive phosphorescent probes based on cellpenetrating peptides. Analytical Biochemistry, 398(1), 24-33.
doi:10.1016/j.ab.2009.10.048.
 Zhuang, X. et al. (2000). Fluorescence quenching: a tool for single-molecule
protein-folding study. PNSA, 97(26), 14241-14244.
 Olmsted, J, & Kearns, D. (1977). Mechanism of ethidium bromide
fluorescence enhancement on binding to nucleic
acids. Biochemistry, 16(16), 3647-3654.
 Atherton, J, & Beaumont P. (1986). Quenching of the fluorescence of
DNA-intercalated ethidium bromide by some transition-metal ions. J. Phys.
Chem., 1986, 90 (10), pp 2252–2259
 Fluorescence resonance energy transfer (fret). (2010). Retrieved from
http://www.andor.com/learning/applications/Fluorescence_Resonance/