Chem 24 FRET Lecture

Chem 24 FRET Lecture - Fluorescence Spectroscopy & FRET...

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absorption fluorescence This energy difference is called the Stokes shift
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What do these Stokes shifts tell you about the nature of the absorption Fluorescence Spectroscopy & FRET
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another form of the Jablonski plot electrons in the S1 state can undergo spin conversion if there is overlap in the excited state energies can be associated with solvent relaxation (10 -10 to 10 -12 sec) relaxation of a polar solvent increases the Stokes shift (lowers the energy of the T 1 state)
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is the fluorescence lifetime of the S 1 (or other fluorescing) state k nr is the non-radiative relaxation rate
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Quenching is any process that decreases the fluorescence intensity Quenching mechanisms: Collisional (dynamic): interactions of a Quencher with the excited state Static: Interactions of a quencher with the ground state
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Collisional quenching: the excited state fluorophore is deactivated upon contact with some other molecule in solution – the Quencher The Quencher must diffuse to the fluorophore during the lifetime of the excited state Upon contact, the fluorophore returns to the ground state without photon emission   Q K Q k F F F F SV q withQ noQ 1 1 0 0 Stern-Volmer Constant [Q] F 0 /F 1 slope = K SV Stern-Volmer Plot
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Collisional Quenching can yield information about the location of a fluorophore on a protein Say we have have used a laser to excite fluorescence from a tryptophan on a protein where is the tryptophan? bottom are variations of Stern-Volmer plots
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Quenchers are things like Oxygen, Bromine, or Iodine (cause fluorophore to cross to a triplet state) Cu 2+ , Pb 2+ , Cd 2+ Electron is donated from the excited state to these ions acrylamide quenches tryptophan, pyrene, ad other fluorphores Fluorescence Spectroscopy & FRET
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Collisional quenching occurs via diffusion of the quencher Consider the root mean square distance over which a quencher can diffuse during an excited state lifetime Q D x 2 2 a tryptophan has a fluorescent lifetime of 4 nanosecond D Q for O 2 is 2.5×10 -8 cm 2 s -1 44 Ȃ is mean square distance Problem: At what concentration of O 2 would you expect 50% of the tryptophan fluorescence to be quenched, assuming every O2/tryptophan collsion is effective at quenching?
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Energy transfer is another example of a fluorescence quenching mechanism
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Forster Resonance Energy Transfer non-radiative energy transfer between molecules with overlapping emission spectra Donor is excited by light Acceptor fluoresces
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Chem 24 FRET Lecture - Fluorescence Spectroscopy & FRET...

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