Workshop advanced light microscopy

Transcription

1 Workshop advanced light microscopy Multi-mode confocal laser scanning microscope Jan Willem Borst Laboratory of Biochemistry Biomolecular Networks MicroSpectroscopy Centre Wageningen

2 Microspectroscopy Centre

3 Microspectroscopy Fluorescence to study biological samples l Sensitive l Specific l Multiparameter l Living cell l Dynamics Applications u Structural properties of protein Ø Ø Ø Ø Ø distances size and shape state of folding ligand binding dynamic properties (local flexibility)

4 Studying protein function by quantitative imaging techniques n Time lapse imaging n High resolution imaging n Co-localization n Protein dynamics n Protein interactions confocal 4Pi microscopy, STED, PALM, Storm Dual color imaging FRAP, FCS FRET, BiFC

5 Confocal set ups present. n Confocal laser scanning microscope (CLSM) l Zeiss LSM510/confocor2 l Leica SP5 n 2-Photon microscope l Fluorescence Lifetime Imaging (FLIM) n Fluorescence Correlation Spectroscope n Multi Mode confocal laser scanning microscope

6 Multi Mode confocal laser scanning microscope Leica SP5

7 Features Leica SP5 Spectral detection Multi color single molecule Hybrid detectors (HyD) Multi dimensional FLIM Argon laser; 458, 477, 488, 514 nm HeNe 543, 633 nm ü White Light Supercontinuum Laser ü Free Tunable excitation nm ü Simultaneous use of 8 wavelengths

8 Localization and dynamics n Fluorescence Recovery After Photobleaching (FRAP) n Fluorescence Correlation Spectroscopy (FCS)

9 Fluorescence correlation spectroscopy (FCS)

10 FCS: Principles FCS autocorrelates relative fluorescence fluctuations G (τ) = I(t) * I(t + τ) <δi(t) * δi(t + τ)> = 1+ <I> 2 <I> 2 1/ N part : Number of molecules Fluorescence Intensity G (τ) τ D = 54 µs Diffusion time Time (s) Tau (ms)

11 FCS for binding studies α-lps scfv-gfp Gram-negative bacteria Outer lipid membrane consisting of lipopolysaccharides Gram-positive bacteria M.A. Hink et al. J. Biol. Chem. 275, (2000)

12 FCS for binding studies scfv-gfp+gram neg. bacteria Normalized autocorrelation 2 scfv-gfp scfv-gfp + Gram pos. bacteria Tau (msec)

13 Monitoring protein interactions in living cells

14 BRI1-GFP/SERK3-mCherry expressed in epidermal root cells BRI1-GFP" SERK3-mCherry" Merge" Bücherl et al., Plant Physiol accepted"

15 A sense of scale FRET Adapted from Molecular Biology of the Cell, 4 th Ed. (2004) Chapter 1

16 What is FRET? Förster Resonance Energy Transfer (FRET) Donor Acceptor Animation created by S. Jähnichen

17 Fluorescence lifetime Relaxation of excited molecule takes place with certain probability based on the decay rates through a number of different (radiative and/or nonradiative) decay pathways intrinsic property of fluorophore" S 1" S 1" S 1" independent of fluorophore concentration" environmental sensitive" S 0" S 0" l è enables detection of molecular interactions" S 0"

18 Donor Fluorescence Lifetime and FRET δ-pulse F(t) = F(0) exp(-t /τ) F(t) τ = 5 ns Time (ns) Donor S 1 k r S 1 k nr k T Acceptor τ D = r S becomes τ = DA k + k k + k + nr S 0 r nr k T

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20 Interaction of Transcription factors FLIM shows a decrease in fluorescence lifetime of the donor (CFP) upon interaction with an acceptor ( YFP)

21 Quantification De Rybel et al. Dev Cell 2013

22 FLIM in live roots BRI1-GFP 1 BRI1-GFP 1+ SERK3-mCherry BRZ BRI1-GFP 1 BRZ+BL BRI1-GFP 1 SERK3-mCherry 1.9 τ (ns) 2.4 IPS: area s interacting population of RKs

23 Time dependent complex formation Fluorescenc lifetime [ps] Interacting pixel [%] tau ip Time [min]

24 Multi Mode confocal laser scanning microscope n Sensitive confocal (multi-color) imaging n Single molecule experiments l Fluorescence fluctuation spectroscopy l Micro-macro timing events n Molecular interactions in vivo l Multi-color (spectral resolved) FLIM l Multidimensional FLIM (x,y,z or x,y,t)

25 Acknowledgements WUR Biochemistry WUR Biochemistry Biomolecular Networks group Catherine Albrecht Dolf Weijers Bert de Rybel Sacco de Vries Ton Visser Christoph Bücherl Wilma van Esse Danny Hamers Antsje Nolles Lisette Nitsch Jan Willem Borst WUR Biophysics Arjen Bader Johannes Holbein Herbert van Amerongen University of East Anglia Sergey Laptenok