B

z 1 1

0,0,0g 0,0,r

d22p

22p

d

1111

Fitting Model for Diffusion in 3DFitting Model for Diffusion in 3D

d

22p

exp τ4

ω D

-12-8exp scm 10 x 2.4 D

-12-8theory scm 10 x 2.3 D

Fit by 3D GaussianModel:

Aragon & Pecora J. Chem. Phys. 64, 1791-1803 (1976)ICS: D 10-8-10-12 cm2s-1

Diffusing Population: 2D SystemDiffusing Population: 2D System

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

Autocorrelation Function Amplitude: Number Density

Simulation: D = 0.01 m2s-1; <N>=10

Diffusing Population: 2D SystemDiffusing Population: 2D System

(s)

0 10 20 30 40

r 11(

0,0

,)

0.000

0.025

0.050

0.075

0.100

D2D = 0.0101 ± 0.0005 m2s-1

B τ

τ 1 (0,0,0)g τ0,0,r

-1

d1111

Best Fit with 2D

Diffusion Model

Simulation: D = 0.01 m2s-1; <N>=10

Flowing Population & Diffusing Population 2DFlowing Population & Diffusing Population 2D

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

Autocorrelation Function Amplitude: Number Density

Simulation: D = 0.01 m2s-1; <N>diff =5 |v| = 0.071 m s-1; <N>flow =5

(s)

0 10 20 30 40

r 11(

0,0

,)

0.000

0.025

0.050

0.075

0.100

Flowing Population & Diffusing Population 2DFlowing Population & Diffusing Population 2D

D2D = 0.010 ± 0.001 m2s-1

|v| = 0.071 ± 0.001 m s-1

Best Fit with 2D Two populationDiffusion & Flow Model

B τ

τ-exp(0,0,0)g

τ

τ 1 (0,0,0)g τ0,0,r

2

ff11

-1

dd1111

Simulation: D = 0.01 m2s-1; <N>diff =5 |v| = 0.071 m s-1; <N>flow =5

2-Photon ICS on Living Cells2-Photon ICS on Living Cells

10 m

CHO Cell: Actinin/EGFP Plated on Fibronectin

Two-photon fluorescence microscopy 7.5 min 37 °C

QuantifyDynamics &Clustering…

2-Photon ICS on Living Cells2-Photon ICS on Living Cells

10 m

(s)

0 50 100 150

r 11(0

,0,

)

0.00

0.05

0.10

0.15

0.20

0.25

Region 1 64x64 pixels

Diffusion & Flow & Immobile3 Populations

D2D = 0.0031 ± 0.0003 m2s-1

|v| = 1.12 ± 0.07 m min-1

10% Immobile

<N>Diff = 2.6 ± 0.2 m-2

<N>flow = 0.33 ± 0.04 m-2

<N>Immobile = 0.34 ± 0.03 m-2

(s)

0 50 100 150

r 11(0

,0,

)

0.000

0.025

0.050

0.075

0.100

0.125

2-Photon ICS on Living Cells2-Photon ICS on Living Cells

10 m

Region 2 128x128 pixels

Diffusion & Immobile2 Populations

D2D = 4.7± 0.3 x10-4 m2s-1

21% Immobile

<N>Diff = 7.6 ± 0.3 m-2

<N>Immobile = 2.0 ± 0.2 m-2

Transport Map for Transport Map for -actinin in a Living Cell-actinin in a Living Cell

10 m Fraction ImmobileFraction DiffusingFraction Flowing

10 min Diffusion Dist.

10 min Flow Dist.

Wiseman et al. Journal of Cell Science 117, 5521-5534, 2004Highlighted in Nature Reviews Mol. Cell Biol. Vol 5, 953, 2004

2P-Image Cross-Correlation Spectroscopy (ICCS)2P-Image Cross-Correlation Spectroscopy (ICCS)

t=0

t=1

t=2

t=n

BiologicalSample

Auto1 Auto2 Cross

t bta

baab i i

) t ,y (x,i t)y, (x,i ,0 ,0r

Temporal Correlation

tbta

bajab i i

t),y ,(xi t)y, (x,i 0 , ,r

SpatialCorrelation

Wiseman et al., J. Microscopy 200, 14-25 (2000)

Temporal Two-photon ICCSTemporal Two-photon ICCS

Actinin - CFP 5 Integrin-YFP

10 m

2-Photon Imaging: 10 min, t = 5s, 3 hr after platingCHO-K1 Cells on 10 g/mL FN Ex. 880 nm Em. 485 & 560 nm

B τ

τ-exp (0,0,0)g

τ

τ 1 (0,0,0)g τ0,0,r

2

ffab

-1

ddabab

2D Diffusion 2D Flow (s)

0 50 100 150

r ab(0

,0,

)

0.0

0.2

0.4

0.6Crosstalk Corrected

Spatio-Temporal Image Correlation SpectroscopySpatio-Temporal Image Correlation Spectroscopy

t t

11 i i

) t ,y ,i(x t)y, i(x, , ,r

Calculate r11(,,) Central Peak is r11(0,0,)

t=0

t=1

t=2

t=3

r11(,,)

(s)

0 10 20 30 40

r 11(0

,0,

)

0.000

0.025

0.050

0.075

0.100

r 11(

0,0

,)

Hebert et al. Biophys. J. 88-3601 (2005)

Full Space Time Correlation on Living CellsFull Space Time Correlation on Living Cells

Directed Flow of -actinin at the basal membrane

Hebert et al. Biophys. J. 88-3601(2005)

Raw Data

ImmobileFiltered

r(0,0,)

Correlation PeakTracking

0 50 100 150 200-1.5

-1.0

-0.5

0.0

0.5

Gau

ssia

n po

sitio

n (

m)

(s)

x position y position

0 200 4000.10

0.15

0.20

r(0,

0,)

Tem

pora

l Cor

rela

tion

Fun

ctio

n

(s)

Temporal Correlation Function Fit (2 pop flow/diffusion)

i)

ii)

Δt=15s 45s 75s 105s 135sA)

B) C)

1 μm

r()

B

τ

τ-expA

τ

τ 1 A τ0,0,r

2

ff

-1

dd11

Vector Maps of Vector Maps of -actinin MEF Cell-actinin MEF Cell

TIRF Microscopy Time 100 s with Images sampled at 0.1 HzDr. Claire Brown and Ben Hebert

Vector Maps of Vector Maps of -actinin-actinin

0 μm/min

9 μm/min

5 μm

0.0 0.2 0.4 0.6 0.8 1.0 1.20.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Re

tro

gra

de

flo

w (

m/m

in)

Protrusion rate (m/min)0 s 2.8 s1.5 s

r()

r()Ff

Inverse relationship b/w retrograde flow & protrusion speed(similar to actin)

Quantum Dots…NanoparticlesQuantum Dots…Nanoparticles

Photostable…Different sizes…Different Colours

Colored Marker Tags for Molecules

CdSe CoreZnS CapSurfaceFunctionalized

Quantum Dot: Semiconductor Materials

10 nm

Silicon &Germanium Based Quantum Dots

Particle Tracking of QD Labeled AMPA ReceptorsParticle Tracking of QD Labeled AMPA Receptors

Richard Naud (Wiseman Group) withProf. Paul DeKoninck Laval University

20 m

Rat Purkinje neuron

Dendritic Spine and Synapse

‘Off’ state

‘On’ state

(CdSe)ZnS – Streptavidin (QD605) (CdSe)ZnS – Streptavidin (QD605) TIRF TIRF Illumination CCD Illumination CCD Detection Detection 50ms Integration Time 50ms Integration Time 2000 Frames 2000 Frames

Nirmal et al. Nature(London) (1996)

But…Quantum Dots Blink!But…Quantum Dots Blink!

See Bachir et al. JAP 99 (2006)Affects ICS measurements

Single Dot i(t) trace

Some New Things: kICSSome New Things: kICS

Point sourcefluorescence

emitters

Imageseries

2D Fouriertransformof images

k-space timecorrelation

function

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Is This Just another Acronym?

No! k-space Correlation has distinct advantages…

For Photobleaching and Blinking of FluorophoresSpecial Thanks to Prof. David Ronis

See Kolin et al. Biophysical Journal 91 3061-3075 (2006)

0 2 4 6 8-15

-10

-5

0

5

10

0 2 4 6 8-101

Residuals

InterceptPhotophysics

SlopeTransport Properties

Some New Things: kICSSome New Things: kICS

Transport CoefficientsIndependent ofPhotophysics

Blinking orPhotobleaching!

Determine the slopes for each value of τ, plot them as a function of τ:

2 4 6 8

-0.12

-0.11

-0.1

-0.09

-0.08

-0.07

-0.06

-0.05

2 4 6 8-0.01

0

0.01

Residuals

Intercept

Slope

Some New Things: kICSSome New Things: kICS

D independentof o

No non-linearCurve fitting

Slope

kICS Live cell measurement kICS Live cell measurement 5 integrin5 integrin

For a given :

ConclusionsConclusions

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!