Polarised light. Polarised light. Polarised light. Polarizer. Polarisation

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1 Polarisation UNIVRSITY OF PÉCS MDICAL SCHOOL Fluorescence anisotrop, FRT In photograph! Miklós Nitrai, Februar 10, 2015 Wh? Polarised light Polarised light Light: lectro-magnetic radiation - a transverse wave (a moving wave that consists of oscillation which is perpendicular to the direction of propagation) Natural or non-polaried light: The electric vector () of the light is moving (vibrate) in all (different) planes. Plane polaried light: The electric vector () of the light is moving (vibrate, oscillate) in a single plane (The movement of the electric vector is arranged in one direction.) Polarised light Polarier An optical device that can convert the unpolaried (mied polariation) electromagnetic wave into a beam with single polariation state. Tpes Beam splitting polariers (prism based polariers) Absorption based polariers (film polariers-sun glasses) 1

2 The principle of polarisers Where did ou use polarisers? Optical activit Polarisation, intensit Optical activit: rotation of the plane of the polaried light. The intensit in different directions: Θ α = α λt lc Ι = Ι ma cos 2 Θ polarier D-fructose L-fructose Parallel: Θ = 0, I = Ima Perpendicular: Θ = 90, I = 0 chiral molecules Polarimetr no ecitation! Photoselection The absorption vector MA = absorption vector θa No absorption (θ= 90o) ecitation Maimal absorption (θ= 0o) 2

3 What happens after the photoselection? Photoselection r r Movements: - translation; - rotation. Within τ time! The emission vector How to describe the rotational motion? M = emission vector IZ θ ecitation Detector The decrease of the component! IZ θ Scheme of a spectrofluorometer light source ecitation monochromator F M P 90o IZ vs. Isum = IZ + + S sample holder P M emission monochromator D detection Vertical or horiontal polarisers: (Compared to the total intensit.) IVV IVH IHV IHH Polarisers in the light paths!!! 3

4 So the total intensit is: Isum = IZ + + IZ θ Isum = IVV + IVH + IVH Fluorescence polarisation Isum = IVV + 2IVH? Fluorescence polariation Fluorescence polariation p = (IVV - GIVH) / (IVV + GIVH) p = (IVV - GIVH) / (IVV + GIVH) Verticall aligned polarier on the ecitation side Horiontall aligned polarier on the emission side If τ = 0: IVV ma., IVH = 0, so p = 1. Correction factor G = IHV / IHH If τ is ver long: IVV = IVH, so p = 0. dimensionless depends on rotational motion of the fluorophore But not additive!!! not additive can change between 0 and 1 mission anisotrop mission anisotrop r = (IVV - GIVH) / (IVV + 2GIVH) Verticall aligned polarier on the ecitation side Horiontall aligned polarier on the emission side G = IHV / IHH dimensionless depends on rotational motion of the fluorophore additive! Remember! Isum = IZ + + Isum = IVV + IVH + IVH Isum = IVV + 2IVH 4

5 What is it good for? can provide information on the rotational mobilit of proteins intermolecular associations can be monitored conformational changes (denaturation) of the proteins can be monitored eamine the internal fleibilit of proteins stud of membranes (viscosit) Applications Describes rotation Polaried light The time dependence of anisotrop τ IZ θ r IZ θ r (t ) = r0 ai ep( t / θ ) I(t) = I0 ep-t/τ Partiall polaried light t The polariation of the emitted light can change because of the rotational diffusion of the moleclue. What does it remind ou? Time-scale of the changes How to measure distances in a molecule? ecitation energ fluorescence phosforescence 10-9 s 10-3 s ecitation Fluorescence Resonance nerg Transfer s (FRT) ground state relaation quenching FRT ecited state internal conversion (heat) 5

6 Fluorescence (or Förster tpe) resonance energ transfer (FRT) - Theodor Förster, 1948 What is the dipol-dipol interaction? Apolar molecule: the charge distribution is homogeneous in the molecule; Polar molecule: the distribution is not homogeneous, the centers of the positive and negative charges are located in the different places; FRT: radiation-less dipol-dipol energ transfer interaction between the ecited donor molecule and an appropriate acceptor molecule. Dipol-molecule : a molecule with two poles. Conditions of FRT A fluorescent donor molecule. The appropriate orientation of the donor and Overlap between the donor emission and acceptor absorption spectra. Distance range of 2-10 nm! absorption or fluorescence intensit What is the spectral overlap? wavelength (nm) FRT The relaation of the donor through its energ transfer interaction with the acceptor! How can we use it to measure distance? hνg D + hνd kt ~ 1/R6 A hνa + R The Jablonski scheme of FRT 6

7 Förster critical distance: R0 FRT efficienc The Förster critical distance is the distance at which the transfer efficienc is 0.5 (50 %). = 1 (FDA / FD) Tpical values: where FDA: donor intensit with the acceptor FD : a donor intensit without the acceptor. Can also be calculated with lifetimes! = 1 (τda / τd) Donor Acceptor Ro (Å) Fluorescein Tetramethlrhodamine 55 IADANS Fluorescein 46 DANS Dabcl 33 Fluorescein Fluorescein 44 BODIPY FL BODIPY FL 57 Fluorescein QSY 7 and QSY 9 des 61 Distance dependence of the FRT efficienc Distance dependence of the FRT efficienc R06 R06 + R 6 Determination of distances! FRT efficienc = Molecular ruler! donor-acceptor distance as R0 units Applications of FRT How to measure FRT distances? The determination of FRT distances To stud the establishment of interactions between molecules; To stud intra-molecular structural changes. 1. Choose an appropriate fluorophore pair! 2. Measure the intensities! 3. Calculate the FRT efficienc! 4. Determine the distance! 7

8 9-anthrolnitrile (ANN) binding to mosin head An eample: 9-Anthrolnitrile binding to mosin head (S1) 12 serine can be the potential binding site for ANN (donor). It was shown that 1 of the 12 serines is binding the fluorophore in S1? Which one? Acceptor binding sites with known positions. 9-anthrolnitrile (ANN) binding to mosin head Summar - polarised light; - photoselection; - fluorescence anisotrop; - FRT; - applications. The fluorophore bound to Ser-181! Thank ou! 8