Fluorescence polarisation, anisotropy FRAP

Transcription

1 Fluorescence polarisation, anisotropy FRAP

2 Reminder: fluorescence spectra Definitions! a. Emission sp. b. Excitation sp.

3 Stokes-shift The difference (measured in nm) between the peak of the excitation and the emission spectrum. - loss of energy within the system!

4 In the case of phosphorescence A type of photo-luminescence. Time-scale: ~ s (~ms - s) Singlet - triplet - singlet transition Forbidden (it can happen but very rarely)!

5 Jablonski diagram; spectra Excited-state Internal conversion or vibrational relaxation heat (10-12 s) S 1 S 1 T 1 : inter-system crossing ( s) T 1 excitation (10-15 s) S 0 S 1 T 1 S 0 ( s ) phosphorescence h h S Vibrational levels Ground-state

6 Phosphorescence spectra

7 Basic fluorescence parameters Intensity Fluorescence spectrum Quantum efficiency Fluorescence lifetime Polarisation

8 Quantum-efficiency (Q) How efficiently will be the absorbed energy converted into the light. Q number of the emitted photons number of theabsorbed photons

9 Basic fluorescence parameters Intensity Fluorescence spectrum Quantum efficiency Fluorescence lifetime Polarisation

10 Fluorescence lifetime ( ) The average length of the excited state of a fluorophore before emitting a photon. 1.2 F F ( kt k nr ) t F0e 0 F e t F e t

11 How to measure fluorescence: the steady-state case

12 The scheme of a fluorimeter F M S 90 o M Non linear arrangement!!! 90 degree between the ingoing and outcoming light D

13 The advantages -great sensitivity and low detection limit -fluorophores are sensitive to the environment

14 Basic fluorescence parameters Intensity Fluorescence spectrum Quantum efficiency Fluorescence lifetime Polarisation

15 Polarisation In photography! Why?

16 Electromagnetic waves

17 Polarization Light: Electro-Magnetic radiation - a transverse wave (a moving wave that consists of oscillation which is perpendicular to the direction of propagation) Natural or non-polarized light: The electric vector (E) of the light is moving (vibrate) in all (different) planes. Plane polarized light: The electric vector (E) of the light is moving (vibrate, oscillate) in a single plane (The movement of the electric vector is arranged in one direction.)

18 Polarizer An optical device that can convert the unpolarized (mixed polarization) electromagnetic wave into a beam with single polarization state. Types Beam splitting polarizers (prism based polarizers) Absorption based polarizers (film polarizers-sun glasses)

19 The principle of polarisers

20 Polarised light

21 Polarised light

22 Where did you use polarisers?

23 Optical activity Optical activity: rotation of the plane of the polarized light lc D-fructose L-fructose t chiral molecules no excitation! Polarimetry

24 Polarisation, intensity The intensity in different directions: polarizer 2 max cos Parallel: Θ = 0, I = I max Perpendicular: Θ = 90, I = 0

25 The absorption vector z A M A = absorption vector E y excitation x

26 Photoselection No absorption ( = 90 o ) Maximal absorption ( = 0 o )

27 E Photoselection

28 What happens after the photoselection? E Movements: - translation; - rotation. Within time!

29 How to describe the rotational motion?

30 The emission vector z M E = emission vector I Z E excitation E I X I Y y Detector x

31 The decrease of the z component! z I X I Z E I Y y I Z vs. I sum = I Z + I X + I Y x (Compared to the total intensity.)

32 The scheme of a fluorimeter F M P S Vertical or horizontal polarisers: 90 o P I VV I VH M I HV I HH D Polarisers in the light paths!!!

33 So the total intensity is: z I sum = I Z + I X + I Y I X I Z E I Y y I sum = I VV + I VH + I VH I sum = I VV + 2I VH x

34 Fluorescence polarisation

35 Fluorescence polarization p = (I VV - GI VH ) / (I VV + GI VH ) Vertically aligned polarizer on the excitation side Horizontally aligned polarizer on the emission side Correction factor G = I HV / I HH dimensionless depends on rotational motion of the fluorophore not additive can change between 0 and 1

36 Fluorescence polarization p = (I VV - GI VH ) / (I VV + GI VH ) If = 0: I VV max., I VH = 0, so p = 1. If is very long: I VV = I VH, so p = 0. But not additive!!!

37 Emission anisotropy

38 Emission anisotropy r = (I VV - GI VH ) / (I VV + 2GI VH ) Vertically aligned polarizer on the excitation side Horizontally aligned polarizer on the emission side G = I HV / I HH dimensionless depends on rotational motion of the fluorophore additive! Remember! I sum = I Z + I X + I Y I sum = I VV + I VH + I VH I sum = I VV + 2I VH

39 Applications

40 Fluorescence emission z z Polarized light I Z E τ I Z E I Y y I Y y I X I X x x Partially polarized light The polarization of the emitted light can change because of the rotational diffusion of the moleclue.

41 The time dependence of anisotropy r I(t) = I 0 exp -t/ r( t) r a exp( t / ) 0 i t What does it remind you?

42 What is it good for? can provide information on the rotational mobility of proteins intermolecular associations can be monitored conformational changes (denaturation) of the proteins can be monitored examine the internal flexibility of proteins study of membranes (viscosity)

43 An example

44 De novo polimerisation of actin Actin monomer Actin filament Depolymerisation Dimer Elongation Nucleation Trimer T. D. Pollard: Cell, 112, , 2003.

45 The domain structure of formins GBD FH3 FH1 FH2 DAD Mammalian Diaphanous-related formins Formin homology (FH) domains: FH1, FH2, FH3 Rho-GTPase binding domain (GBD): Diaphanous Autoregulatory domén (DAD): Autoregulation

46 Question What is the effect of actin on the structure of the actin filament?

47 Methods Protein preparations (formin, myosin, actin, tropomyosin). Anisotropy measurements.

48 The principals twisting bending monomer rotation segmental motion

49 Time dependence of anisotropy longer rotational correlation time (ns) shorter rotational correlation time (ns) [mdia1-fh2] ( M) A [mdia1-fh2] ( M) flexibility (arb. units) 3,0 T = 30 o C 2,5 2,0 1,5 1,0 0,0 0,2 0,4 0,6 0,8 1,0 [mdia1-fh2+linker] : [actin] The formins increased the flexibility of actin filaments.

50 How to understand it? Side - binding 3,0 T = 30 o C End - binding flexibility (arb. units) 2,5 2,0 1,5 1,0 0,0 0,2 0,4 0,6 0,8 1,0 [mdia1-fh2+linker] : [actin]

51 Further parameters and correlations

52 Limiting anisotropy (r 0 ) For a frozen fluorophore. r cos 1 2 z Absorption vector Emission vector : the angle between the absorption and emission vectors. y r x

53 What can influence the limiting anisotropy? - Rotational motion; - Fluorescence lifetime!

54 Francis Perrin ROTATIONAL DIFFUSION depolarization of the emitted light Perrin equation: Limiting anisotropy r r 0 r D Diffusion coefficient Rotational correlation time (the time period that can be correlated with rotational diffusion of a molecule) V RT Stokes-Einstein relationship

55 Perrin equation r is inversely or linearly proportional to the different parameters. r ~ r r r 0 6D r 0 1/ 1/D

56 FRAP

57 Fluorescence recovery after photobleaching /FRAP/

58 Photobleaching Irreversible photochemical destruction of the fluorophor ( burn out ) Excitation light destroy the fluorescent molecules How to avoid: anti-photobleaching conditions (eg. glucose oxidase catalase mercaptoethanol coctail) decrease exposition time pulsatile excitation low intensity excitation light long-life fluorofor (eg. Alexa) How to use: eliminate background autoquenching FLIP, FRAP

59 Relative fluorescence (%) Method 1. Fluorescent sample 2. Area is bleached out (eg. with laser) Photobleach Lippincott-Schwartz, Fluorescence recovery I 0 4. New equillibrium (usually: y < x) I0 2 x y 0 t 1/2 Time

60 Practical use Lateral diffusion of membrane components Monomer turnover Protein motility Determination of diffusion coefficient D 2 4t 1/2