Feb. 24 Presentation FRET & Fluorescence
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Transcript Feb. 24 Presentation FRET & Fluorescence
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/