Fitting Model for Diffusion in 3 D Fit

Fitting Model for Diffusion in 3 D Fit by 3 D Gaussian Model: ICS: D 10 -8 -10 -12 cm 2 s-1 Aragon & Pecora J. Chem. Phys. 64, 1791 -1803 (1976)

Diffusing Population: 2 D System Simulation: D = 0. 01 m 2 s-1; <N>=10 Autocorrelation Function Amplitude: Number Density Simulation: <N>=10 <N>spatial = <g(0, 0)n>-1 = 10. 18 ± 0. 03 <N>temporal = g(0, 0, 0)-1 = 10. 18 ± 0. 01

Diffusing Population: 2 D System Simulation: D = 0. 01 m 2 s-1; <N>=10 D 2 D = 0. 0101 ± 0. 0005 m 2 s-1 Best Fit with 2 D Diffusion Model

Flowing Population & Diffusing Population 2 D Simulation: D = 0. 01 m 2 s-1; |v| = 0. 071 m s-1; <N>diff =5 <N>flow =5 Autocorrelation Function Amplitude: Number Density Simulation: <N>diff =5 <N>flow =5 <N>spatial = <g(0, 0)n>-1 = 10. 01 ± 0. 03 <N>temporal : <N>diff =4. 9 ± 0. 3 <N>flow =5. 1 ± 0. 3

Flowing Population & Diffusing Population 2 D Simulation: D = 0. 01 m 2 s-1; |v| = 0. 071 m s-1; <N>diff =5 <N>flow =5 D 2 D = 0. 010 ± 0. 001 m 2 s-1 |v| = 0. 071 ± 0. 001 m s-1 Best Fit with 2 D Two population Diffusion & Flow Model

2 -Photon ICS on Living Cells CHO Cell: Actinin/EGFP Plated on Fibronectin Quantify Dynamics & Clustering… 10 m Two-photon fluorescence microscopy 7. 5 min 37 °C

2 -Photon ICS on Living Cells 10 m D 2 D = 0. 0031 ± 0. 0003 m 2 s-1 |v| = 1. 12 ± 0. 07 m min-1 Region 1 64 x 64 pixels 10% Immobile Diffusion & Flow & Immobile 3 Populations <N>Diff = 2. 6 ± 0. 2 m-2 <N>flow = 0. 33 ± 0. 04 m-2 <N>Immobile = 0. 34 ± 0. 03 m-2

2 -Photon ICS on Living Cells 10 m D 2 D = 4. 7± 0. 3 x 10 -4 m 2 s-1 Region 2 128 x 128 pixels 21% Immobile Diffusion & Immobile 2 Populations <N>Diff = 7. 6 ± 0. 3 m-2 <N>Immobile = 2. 0 ± 0. 2 m-2

Transport Map for a -actinin in a Living Cell 10 m Fraction Immobile Fraction Diffusing Fraction Flowing 10 min Diffusion Dist. 10 min Flow Dist. Wiseman et al. Journal of Cell Science 117, 5521 -5534, 2004 Highlighted in Nature Reviews Mol. Cell Biol. Vol 5, 953, 2004

2 P-Image Cross-Correlation Spectroscopy (ICCS) Wiseman et al. , J. Microscopy 200, 14 -25 (2000) Biological Sample Auto 1 Auto 2 Cross Spatial Correlation t=0 t=1 t=2 t=n Temporal Correlation

Temporal Two-photon ICCS CHO-K 1 Cells on 10 g/m. L FN Ex. 880 nm Em. 485 & 560 nm 2 -Photon Imaging: 10 min, dt = 5 s, 3 hr after plating Crosstalk Corrected Actinin - CFP 10 m 5 Integrin-YFP 2 D Diffusion 2 D Flow

Calculate r 11(x, h, t) Central Peak is r 11(0, 0, t) t=0 t=1 r 11(0, 0, t) Spatio-Temporal Image Correlation Spectroscopy t r 11(x, h, t) t=2 h t=3 Hebert et al. Biophys. J. 88 -3601 (2005) x

Full Space Time Correlation on Living Cells Directed Flow of -actinin at the basal membrane A) i) Δt=15 s 45 s 75 s 105 s 135 s Raw Data Immobile Filtered r(x, h, t) ii) 1 μm B) C) r(0, 0, t) t Hebert et al. Biophys. J. 88 -3601 (2005) Correlation Peak Tracking t

Vector Maps of a-actinin MEF Cell TIRF Microscopy Time 100 s with Images sampled at 0. 1 Hz Dr. Claire Brown and Ben Hebert

Vector Maps of a-actinin 9 μm/min 0 μm/min 5 μm Inverse relationship b/w retrograde flow & protrusion speed (similar to actin) r(x, h, t)Ff 0 s 1. 5 s 2. 8 s

Quantum Dots…Nanoparticles Photostable…Different sizes…Different Colours Quantum Dot: Semiconductor Materials 10 nm Cd. Se Core Zn. S Cap Surface Functionalized Silicon &Germanium Based Quantum Dots Colored Marker Tags for Molecules

Particle Tracking of QD Labeled AMPA Receptors 20 m Dendritic Spine and Synapse Rat Purkinje neuron Richard Naud (Wiseman Group) with Prof. Paul De. Koninck Laval University

But…Quantum Dots Blink! (Cd. Se)Zn. S – Streptavidin (QD 605) TIRF Illumination CCD Detection 50 ms Integration Time 2000 Frames See Bachir et al. JAP 99 (2006) Affects ICS measurements ‘On’ state Single Dot i(t) trace ‘Off’ state Nirmal et al. Nature(London) (1996)

Some New Things: k. ICS Point source fluorescence emitters Image series 2 D Fourier transform of images k-space time correlation function Is This Just another Acronym? No! k-space Correlation has distinct advantages… For Photobleaching and Blinking of Fluorophores Special Thanks to Prof. David Ronis See Kolin et al. Biophysical Journal 91 3061 -3075 (2006)

Some New Things: k. ICS Intercept Photophysics Slope Transport Properties Transport Coefficients Independent of Photophysics Blinking or Photobleaching! Residuals

Some New Things: k. ICS Determine the slopes for each value of τ, plot them as a function of τ: Intercept Slope D independent of wo No non-linear Curve fitting t Residuals

k. ICS Live cell measurement a 5 integrin Slope For a given :

Conclusions Fluctuations Contain Information about Molecules Fluctuation Size…Concentrations/Oligomerization Fluctuation Time…Dynamics/Kinetics FCS…Temporal Analysis of Fluctuations Image Correlation…Space & Time Analysis Quantum Dots…Promising…but not perfect!