Post on 25-Dec-2015
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
Ground 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)
FRAP
Fluorescence Recovery After Photo-bleaching
Used to examine Brownian motion and
2-D interactions in membranesExamine 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
Fluorescence Quenching
Environmental effect Solvent Additional solutes Other moieties
Drastically effects quantum yield as well as rate of fluorescence
How does it work?Fluorophore
Molecular Oxygen
Fluorophore
Molecular Oxygen
Fluorescent Not
Fluorescent
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:
Distance between fluorophores (r)
r0= Distance where efficiency equal 0.5
D
ADEff
1
660
60
rr
rEff
Phosphorescence
Emission of light resulting from quantum-mechanically forbidden transitions
“Glow in the dark”
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 cell-penetrating 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/