Fluorescence Correlation

Transcription

1 Theory setions of the leture Studying dynami proesses in ells and model membranes with Fluoresene Correlation Spetrosopy () and FCS-urves LSM (3D projetion) Confoal Laser Sanning Mirosopy presented at VW summershool Complex Materials Intern. Univ. Bremen, June Kirsten Baia and Petra Shwille Dept. of Biophysis, Tehnishe Universität Dresden

2 Fluoresene Correlation Spetrosopy (FCS) two-photon exitation Intrinsi (auto-)fluoresene is too weak (Trp et.) speifi labelling with strong fluorophores (fluoresent proteins, hemial dyes, quantum dots) 488 nm Absorption and Emission (relative units) nm Absorption and Emission (relative units) nm Absorption and Emission (relative units) λ/nm λ/nm λ/nm

3 Measuring diffusion: Fluoresene Correlation Spetrosopy Confoal mirosope setup Exitation profile Laser intensity axial lateral Detetion profile Detetion effiieny ω o = 0.2 µm V eff = 0.2 fl axial Pinhole lateral

4 Confoal setup small detetion volume illumination: NA, λ, beam diameter smaller detetion: Exitation: Fous angle δ Main beam splitter (HFT) Detetion: Sample Detektion angle α Objetive Parallel Laserlight to detetor Pinhole Optis Pinhole Optial Fiber z high NA objetive pinhole APD Detetor Software-Correlation Pinhole largely rejets fluoresene from above/below the foal plane.

5 Confoal imaging and onfoal FCS Confoal imaging (LSM) y F Fluoresene intensity F, Position (x,y) Autoorrelation () x FCS δf(t) N ( ) = F( t) F( t + ) F( t) 2 diff Fixed fous position t Fluoresene flutuations δf(t) orrelation time

6 The autoorrelation funtion fluoresene F(t) F(t+) F(t+) F(t+) F(t+) t t+ t+ orrelation time averaging over time t () time axis t ( ) = F( t) F( t F( t) + ) 2

7 Interpreting FCS urves <F> F / khz () time / s blinking 60 z o ω o Non-linear least-squares fitting of a model equation, e.g. () = N + 2 diff ω + z 0 0 diff /Ν diff z o /ω o = struture parameter ( F trip + F trip exp( to the experimental FCS urve. f )) + 0 baseline (0 or ) on log sale Important parameters: Mobility: D = ω o ² / (4 diff ) Conentration: = N / V eff Partile brightness: η = <F> / N

8 Model equations for Model equations for different types of motion different types of motion = 2 0 exp ) ( ω v diff + ) = ( diff diff S ² ) ( + + = α + = anom ) ( transport 3D-diff. 2D-diff. anom. diff.

9 Summary: Fluoresene flutuations Origins: diffusion; flikering (triplet, isomerization, protonation...) Prerequisites: low onentrations (nm( nm), small measurement volume (onfoal setup, TIRF setup) bright & photostable fluorophores, Information: mobility (diffusion oeff., veloity) `flikering` (relaxation time) onentration partile brightness mobility (at least D= m²/s)

10 Summary: Autoorrelation normalized to show diffusion K. Baia, S.A. Kim, P. Shwille, Nat. Meth., 2006

11 FCS assesses a wide range of dynamis (), normalized diff / ms D / (m²/s) (A) (B) (C) (D) (E) A B C D E (A) Alexa 488 (B) efp in buffer (C) efp protein (D) CaMKII-eFP protein in the ytoplasm (E) fluoresent lipid analogue (dii) in the plasma membrane 3D 3D 2D.0 E / ms (C) efp in HEK ell (D) CaMKII-eFP in HEK ell (E) dii in HEK ell plasma membrane Baia K, Shwille P, Methods 2003 (Review)

12 How to determine moleular interations? Coloalization??? FRET FCS diffusion analysis normalized (),0 0,8 0,6 0,4 0,2 0,0 0,0 0, [ms] 0 Moleular mobility

13 Limitations of fluoresene autoorrelation for binding analysis multiple omponents only resolved, if signifiantly different diffusion oeff. D.5 diff / D in solution: D / M /3 (), normalized ligand in solution 0.0 E inreasing fration of free fluoresent ligand in membranes: Saffman-Delbrük D / (0-8 m²/s) R / nm in ells: variety of interations

14 More information from dual-olor ross-orrelation orrelation K. Baia, S.A. Kim, P. Shwille, Nat. Meth., 2006 (Review)

15 Setup for dual-olor FCCS images by Tobias Kohl and Mihael Jahnz

16 Dual-olor FCCS (0) = V eff R R, t, t η R R η R η R η Ideally: red speies observed only in red hannel, green speies observed only in green hannel, double-labeled speies in both hannels r ( 0) = V eff R, t R =, t r (0) ( 0) g ( 0) = V eff R = R, t g, t (0) ( 0)

17 Summary: dual-olor FCCS K. Baia, S.A. Kim, P. Shwille, Nat. Meth., 2006 (Review)