4 Single molecule FRET

Transcription

1 4 Single molecule FRET

2 FRET basics Energie Dipole-dipole interaction Teil I SM Fluo, Kap. 4 FRET

3 FRET basics transfer rate (from Fermis Golden Rule) k t = r 6 apple 2 9 ln(10) n 4 N A Z d f D ( )" A ( )/ 4 0 : fluorescence lifetime donor r : donor acceptor distance we d like to know apple 2 : orientation factor n : refractive index Z d f D ( )" A ( )/ 4 : overlap integral Teil I SM Fluo, Kap. 4 FRET

4 Overlap integral Z d f D ( )" A ( ) 4 Teil I SM Fluo, Kap. 4 FRET Wellenlänge (nm)

5 Förster radius disctance, where transfer rate equals sum of all other rates k t = r 6 k t = 1 0 r0 r apple 2 9 ln(10) n 4 N A Z d f D ( )" A ( )/ 4 r 6 0 Teil I SM Fluo, Kap. 4 FRET

6 Transfer efficiency ratio of transferred quanta to absorbed quanta Can be measured Via emission from donor and acceptor E t = A D + Via fluorescence lifetime of donor (Transfer = additional relaxation channel!) A E t =1 D,t D,0 Teil I SM Fluo, Kap. 4 FRET

7 Transfer efficiency distance dependent 1/2 at r=r Transfereffizienz E t = r r Teil I SM Fluo, Kap. 4 FRET Donor-Akzeptor-Abstand (Å)

8 4.1 Orientation factor apple = 2 cos D cos A sin D sin A cos apple 2 =1 apple 2 =0 How to distinguish orientation and distance? Teil I SM Fluo, Kap. 4 FRET, 4.1 Orientierung

9 Rotational averaging Dynamic mean (Both dipoles rotate fast compared to all relaxation rates): <κ 2 >=2/3 One dipole is rotating fast, the other one slowly: κ 2 2/3 Static ensemble average, slow rotation: κ 2 =0,476 Teil I SM Fluo, Kap. 4 FRET, 4.1 Orientierung

10 Fluorescence anisotropy Determination of rotational freedom Steady state I VH r = I VV I VV +2I VH r=0 for free rotation r=r0 (r0: intrinsic anisotropy, for our dyes 0,4) for no rotation Excite vertical I VV I VH Filters, lenses Teil I SM Fluo, Kap. 4 FRET, 4.1 Orientierung

11 Time resolved anisotropy r(t) = I VV(t) I VH (t) I VV (t)+2i VH (t) = r 0 exp( τ: rotational correlation time t/ ) Teil I SM Fluo, Kap. 4 FRET, 4.1 Orientierung

12 Dye on protein Model 1: Rotation in a cone r(t) = (r 0 r 1 ) e t/ eff + r 1 e t/ M τeff: effective rotation time within cone τm: rotation time of protein r : anisotropy for non-rotating protein Teil I SM Fluo, Kap. 4 FRET, 4.1 Orientierung

13 Dye on protein Model 2: Transient sticking of the dye on protein r(t) =r 0 ye t/ F (y 1)e t/ M y: portion of freely rotating dye Teil I SM Fluo, Kap. 4 FRET, 4.1 Orientierung

14 4.2 Photo physics/chemistry What happens in a FRET system if donor and/or acceptor enter triplet or other dark state? How can dark states like triplet states be avoided? Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie

15 Donor in triplet state No absorption, therefore no emission: donor and acceptor become dark Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie

16 Acceptor in triplet state Everything can happen: If no FRET is occuring: donor fluorescence is increasing Energy transfer to T1 - TN transition of acceptor: Depending on efficiency of this transfer compared to standard FRET S0 - S1 fluorescence of donor can increase, decrease, or remain constant Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie

17 Singlet triplet annihilation Förster-type energy transfer to T1 - TN transition S 3 S 3 T 3 T 2 T 1 S 2 S 1 T 3 T 2 T 1 S 2 S 1 S 0 S 0 Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie

18 Triplet modification Heavy atoms: strong spin orbit coupling, enhance intersystem crossing (fwd and backwd) Triplet quencher (electron exchange) Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie

19 Oxygen as triplet quencher Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie

20 Radical ions - ROXS Radical ionic states - long lived dark states Reducing Oxidizing System (ROXS) depopulates those states Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie

21 Ox ROXS effect Red Roxs Teil I SM Fluo, Kap. 4 FRET, 4.2 Photophysik/-chemie