FLIM Fluorescence Lifetime IMaging

Transcription

1 FLIM Fluorescence Lifetime IMaging

2 Fluorescence lifetime t I(t) = F0 exp( ) τ 1 τ = k f + k nr k nr = k IC + k ISC + k bl Batiaens et al, Trends in Cell Biology, 1999 τ τ = fluorescence lifetime (~ns to ps) (average residence time of the molecule in the excited state) k f = radiative decay rate (photon emission) k nr = nonradiative decay rate Note: quantum yield Q = k r kr + k nr

3 Fluorescence lifetime nr nr From Michael Liebling, UCSB

4 FLIM measurement Batiaens et al, Trends in Cell Biology, 1999

5 FLIM to probe the fluorophore local environmnent FLIM: non-intensity based chemical sensing! τ sensitive to local environment Decay rate can be measured for each pixel of the image Applications: - Contrast between fluorophores with different decay rates (even if same emission wavelength). - Fluorophore local environement - FLIM combined with FRET Suhling et al, Photochem. Phtobiol. Sci., 2005

6 FLIM to probe the fluorophore local environmnent τ sensitive to local environment Suhling et al, Photochem. Phtobiol. Sci., 2005

7 FLIM to characterize FRET FLIM: non-intensity based chemical sensing! Decay rate can be measured for each pixel of the image Applications: - Contrast between fluorophores with different decay rates (even if same emission wavelength). - Fluorophore local environement - FLIM combined with FRET Suhling et al, Photochem. Phtobiol. Sci., 2005

8 Beyond Jablonski: decay rate R: distance between fluorophores R 0 : Foster radius

9 FLIM to characterize FRET FRET decreases the fluorescence lifetime of the donor! Suhling et al, Photochem. Phtobiol. Sci., 2005

10 FLIM to characterize FRET FRET decreases the fluorescence lifetime of the donor! Batiaens et al, Trends in Cell Biology, 1999

11 FLIM Advantages FLIM: non-intensity based chemical sensing! τ is independent of chromophore concentration τ is directly dependent upon excitation-state reactions

12 FRAP Fluorescence Recovery After Photobleaching

13 Mobile fraction and diffusion constant via FRAP Lippincott-Schwarz et al, Nature Review in Molecular and Cell Biology, 1999

14 Diffusion theory

15 Mobile fraction and diffusion constant via FRAP A C A B C B The diffusion coefficient obtained from the recovery curve (depend on the bleaching geometry) See Axelrod et al, Biophysical Journal, 1976 H2A-GFP: Diffusion coefficient ~0.01 μm 2.sec -1

16 FRAP Control 1: linearity between [fluorophore] and signal if linear f 0 : fluorescence signl at start C 0 : unbleached fluorophore concentration at start n: bleach step γ: fraction of fluorophore bleached at each step

17 FRAP Control 2: bleaching during acquisition Fluorescence signal in an unbleached region during the time of acquisition. Good: no significant change in fluorescence level

18 FRAP Control 3: production of GFP during the acquisition time A large area is bleached. Good: no recovery = no GFP production

19 FRAP exemple

20 FCS Fluorescence Correlation Spectroscopy

21 FCS: spontaneous intensity fluctuations Molecular dynamics F(t) Fluorescence intensity fluctuations Number in focus fluctuates From Paul Wiseman, McGill University t From

22 Fluctuation Magnitudes & Fluctuation Times F(t) τ f = Characteristic Fluctuation Time <F>= δf(t) = F(t) - <F> Fluctuation Magnitude t

23 FCS: Fluctuations & Dynamics Fast Dynamics Short τ f Focal Volume 1 μm 3

24 FCS: Fluctuations & Dynamics Slow Dynamics Long τ f Focal Volume 1 μm 3

25 Fluctuation analysis: the auto-correlation function

26 Fluctuation analysis: the auto-correlation function Normalized auto-correlation function:

27 Fluctuation analysis: the auto-correlation function From

28 Fluctuation analysis: the auto-correlation function

29 FCS: characterizing particle motion From

30 FCS: probing intramolecular dynamics diffusion blinking From

31 Colocalization vs true molecular interaction 4 2 Gaussian Beam Focus Optical Microscopy Dynamics at the Price of Spatial Resolution 0-2 ~ 500 nm Truly Interacting Species Dance Partners Versus Simply Colocalized Optical Resolution ~ λ/2 Macromolecules ~ λ/50 From Paul Wiseman, McGill University

32 FCS: dual-color cross-correlation inter-molecular interaction 2 fluorophores: cross- instead of auto-correlation Normalized cross-correlation function: 1 2 cross 1 2

33 FCS: dual-color cross-correlation inter-molecular interaction Dual color FCS can reveal truly interacting of molecules! From

34 FCS: dual-beam cross-correlation measuring velocity R: distance between the 2 focal volumes From

35 FCS: conclusions Non-intensity based technique Sensitive to fluorescence fluctuations High temporal/spatial resolution Measure of : - mobility constant - intra-molecular dynamics - inter-molecular dynamics NOTE: this technique requires a low concentration of fluorophore! Each molecule has to contribute substantially to the measured signal in order to generate fluorescence fluctuations