Structured illumination microscopy

Transkript

1 Structured illumination microscopy scanning periodic excitation over sample periodic image uniform image y min λ λ = = 4nsinΘ 4N.A. two-fold enhancement in resolution

2 Confocal microscope max. lateral resolution with deconvolution λ λ = 2nsinΘ 2N.A.

3 Improving axial resolution: 4π-Microscopy n λ λ 1 cos 2n ( Θ)

4 Optical saturation microscopy Photobleaching S 1 Excitation T 1 Fluorescence Emission S 0

5 Optical saturation microscopy

6 STimulated Emission Depletion Microscopy

7 STimulated Emission Depletion Microscopy excitation laser PSF STED laser

8 STimulated Emission Depletion Microscopy

9 STimulated Emission Depletion Microscopy

10 2-focus Fluorescence Correlation Spectroscopy (2fFCS)

11 Confocal fluorescence microscopy

12 Fluorescence intensity fluctuations

13 Structure of an autocorrelation curve

14 Example: Measured FCS curves of yellow fluorescent protein

15 Amplitude of an autocorrelation curve

16 Normalized amplitude of an autocorrelation curve

17 Ideal molecule detection function Molecule detection function (1/e 2 isosurface) NA = 1.2 wd = 3 mm tubelens = 180 mm n 0 = 1.33 λ ex = 635 nm ω = 4.9 mm focus pos. = 10 μm λ em = 670 nm magn. = 60 pinhole radius = 50 μm

18 Cover-slide thickness deviation

19 Refractive index mismatch

20 Optical saturation

21 2-focus confocal system

22 2-focus confocal system

23 2-focus confocal system

24 Time-tagged time-resolved mode of photon counting Fluorescence decay curve Frequency Laser pulse Data: t 1 t t t t t t t τ τ τ τ τ τ τ τ Decay time (ns)

25 Pulsed Interleaved Excitation (PIE) A B

26 2-focus FCS (2fFCS): two mutually shifted detection volumes

27 2fFCS of diffusion in lipid bilayers: Model systems Black lipid membranes Supported bilayer Giant unilamellar vesicle

28 2fFCS of lipid diffusion in Giant Unilamellar Vesicles

29 2fFCS of lipid diffusion in Giant Unilamellar Vesicles

30 2fFCS of lipid diffusion in Giant Unilamellar Vesicles Giant Vesicles made of DOPC labeled 1 : 400,000 with Atto655-DOPE room temperature inside vesicle water glucose glucose sucrose sucrose outside vesicle diffusion coefficient of lipid water (8.0 ± 0.4) 10-8 cm² / s glucose (7.5 ± 0.4) 10-8 cm² / s 100mM KCl, 30mM Hepes ph 7.2, 1mM CaCl (9.0 ± 0.4) 10-8 cm² / s sucrose (6.8 ± 0.4) 10-8 cm² / s 100mM KCl, 30mM Hepes ph 7.2, 1mM CaCl (7.3 ± 0.4) 10-8 cm² / s

31 2fFCS in solution

32 2fFCS of Atto655 in GdHCl: refractive index dependence ( ) D = ± C cm s

33 2fFCS: optical saturation dependence of Cy5

34 Ca 2+ -binding of Calmodulin: Hydrodynamic radius

35 Ca 2+ -binding of Recoverin

36 Protein folding/unfolding: Tryptophan cage