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Fluorescence Correlation & Image Correlation Methods

Fluorescence Correlation & Image Correlation Methods. Paul Wiseman Department of Physics Department of Chemistry McGill University Montreal, Canada. Overview for Tutorial. Optical Microscopy Dynamics vs. Resolution Fluorescence Correlation Spectroscopy (FCS)

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Fluorescence Correlation & Image Correlation Methods

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  1. Fluorescence Correlation & Image Correlation Methods Paul Wiseman Department of Physics Department of Chemistry McGill University Montreal, Canada

  2. Overview for Tutorial • Optical Microscopy Dynamics vs. Resolution • Fluorescence Correlation Spectroscopy (FCS) • Image Correlation Spectroscopy (ICS) • Image Cross-Correlation Spectroscopy (ICCS) • Spatio-Temporal Image Correlation Spectroscopy • Reciprocal Space Image Correlation Spectroscopy

  3. z r J0(r) or J0(r)2 r J1(r) or J1(r)2 q1 r Optical Resolution Bessel Function of Order zero and its Square Airy Disk in Focal Plane (cross section of PSF) 3D PSF Bessel Function of Order one and its Square

  4. Rayleigh Resolution Criterion Circular Aperture Image~ {J1(x)/x}2 Object Point~ d(x) Ultimate Goal of Microscopy Resolve to closely separate Point sources from the object plane Within the image plane D Obj. Lens d Rayleigh Resolution Criterion q1~1.22 f/(lD)

  5. Truly Interacting Species Dance Partners Diffraction Limited Optical Resolution… Optical Microscopy Dynamics at the Price of Spatial Resolution Gaussian Beam Focus Versus Simply “Colocalized” ~ 500 nm Optical Resolution ~ l/2 Macromolecules ~ l/50

  6. Goal: Measure the Biomolecular Dance 170 mm Optical Microscopy Dynamics…At the price of Limited Spatial Resolution Fluorescence Micrscopy Specificity Low Detection Limits (single Molecule) Paxillin-dsRed (red) & a-actinin GFP (green) in CHO Cell TIRF Microscopy Total time = 50 min dt =15 s

  7. i(t) t Fluctuation Magnitudes & Fluctuation Times Fluorescence Correlation Spectroscopy (FCS) Obj. Lens Elson and Magde ; Magde, et al.Biopolymers (1974) 13, 1-27 ; 29-61

  8. f = Characteristic Fluctuation Time i(t) = i(t) - <i> Fluctuation Magnitude Fluctuation Magnitudes & Fluctuation Times i(t) <i>=26.36 t

  9. FCS: Fluctuations & Dynamics Fast Dynamics Short f Focal Volume  1 mm3

  10. FCS: Fluctuations & Dynamics Slow Dynamics Long f Focal Volume  1 mm3

  11. FCS Instrumentation M1 Sample Laser Dichroic M2 BE Pinhole Photon Detector APD AMP Mirror Computer Filters Temporal ACF Signal Autocorrelator

  12. Correlation Function Amplitude: g(0) Number Density <N> per Beam Area Aggregation State Correlation Function Decay: Mi Fluctuation Relaxation Terms Transport and Kinetics Properties Correlation Function Decay Model: 2D For 2D System; Laser TEM00 Mode ai= Qi/Q1 Ratio of Fluorescent Yields Sum over all fluorescent species N. L. Thompson; Topics in Fluorescence Spectroscopy (1991) 1, 337-378

  13. t=0 t=1 t=2 t=3 t=4 Image Correlation Spectroscopy 100fs, 780-920nm pulse 82MHz rep-rate M1 TiSapph. laser Sample PMT 1 PMT 3 PMT 2 Em. Filters Filter Dichroic mirror M4 Dichroic mirrors M2 +L1 pinhole+L2 M3

  14. Laser TIRF Microscopy ~ 100 nm z depth of field Sample NA 1.45 Obj. Lens Laser Beam Fluorescence Dichroic & Em. Filter CCD Camera ND Filters

  15. Slow or Static Distributions? Receptor Occupation Number Varies across the Membrane Intensity Fluctuations Laser Beam Rasters across Sample <(i)2>/<i> 2 = 1/<N> Mean Number of “Independent” Clusters per Beam Area

  16. White Noise Spatial AC Function A Confocal Image Spatial Image Correlation Spectroscopy Petersen et al. Biophys. J. 65, 1135-1146 (1993); Wiseman and Petersen, Biophys. J. 76, 963-977 (1999)

  17. } h lag variable pixel shift in y } Image i(x,y) x lag variable pixel shift in x Spatial ACF r11(x,h) Correlate Image With Itself Correlation Function Mathematical Correlation Of Image with Itself Spatial Autocorrelation Function (ACF)

  18. * Image i(x,y) F {i(x,y)} F {i(x,y)} F {i(x,y)}* Spatial ACF r11(x,h) Inverse FFT Normalization FFT complex conjugate multiplication Spatial Autocorrelation Function (ACF) Power Spectrum

  19. White Noise Spatial AC Function A Confocal Image Spatial Image Correlation Spectroscopy Petersen et al. Biophys. J. 65, 1135-1146 (1993); Wiseman and Petersen, Biophys. J. 76, 963-977 (1999)

  20. Gaussian Fitting Function Gaussian Fitting Function <N> Independent Fluorescent Entities; Aggregation Nonlinear Least Squares Fitting

  21. t=0 Decay Transport Dynamics Diffusion Coeff. & Flow Speeds Temporal ACF t=1 t=2 t=3 t=n Image Correlation Spectroscopy (ICS) Temporal Autocorrelation of di(x,y,t) = i(x,y,t) - <i> Through Time Series Offset Immobile Population Srivastava and Petersen Methods Cell Sci. 18, 47-54 (1996)

  22. t=0 t=1 t=2 t = 0 t=3 t = 4 t = 1 t = 3 t = 2 t=4 t = n t=n Temporal ICS How to Calculate Normalized Fluctuation Autocorrelation Function Time Lag t = 0 Time Lag t = 1 Time Lag t = 2 Time Lag t = 3 Time Lag t = 4 Time Lag t = n

  23. 5m 3D Diffusion Model • 0.2 m blue fluorescent spheres in sucrose/water solutions • Temperature  21C, 0% sucrose, 2P Microscopy 30f/s Wiseman et al. J. Microscopy 200, 14-25 (2000)

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