Mapping protein dynamics in living cells i fl fl t ti & l tivia fluorescence fluctuation & correlation

analysis in space, time & reciprocal space

Paul W. Wiseman McGill UniversityDept. of Physics, Dept. of Chemistry

SHG I i

Wiseman Lab Biophysical Research McGillWiseman Lab Biophysical Research McGillSHG ImagingHeart Collagen

THG Imaging ofMalaria Hemozoin

Guiding Neurons on Patterned Protein Gradients Guiding Neurons on Patterned Protein Gradients

IKVAV peptide Gradient

Guided

With D S ti C t ti U d M t l

Chick Dorsal RootGanglion onIKVAV peptide gradient

With Dr. Santiago Costantino U de MontrealDr. Tim Kennedy Montreal Neurological InstituteBelisle et al. Lab on a Chip in press

Goal: Measure the Biomolecular DanceGoal: Measure the Biomolecular Dance

170 mWhat are rules of the game withinthe Living Cell?the Living Cell?

Physico-chemical Understanding

New Techniques are needed…

Paxillin dsRed (red) &

i) Spatio-temporal Image Correlation Spectroscopy (STICS) Paxillin-dsRed (red) &

-actinin GFP (green)in CHO CellTIRF Mi

ii) k-space Image Correlation Spectroscopy (kICS) TIRF Microscopy

Total time = 50 min t =15 s

Spectroscopy (kICS)

How do We Observe the Dance? How do We Observe the Dance? FluorescenceFluorescenceG Fl t P t i ti f i t t t tGreen Fluorescent Protein genetic fusion construct to create a Protein with a fluorescent protein attachedExcite GFP with focused laser light…Collect Fluorescence photons

Emitting

g p

g

~ 3 nmPhotobleachedex 488 nm

em 509 nmFrom http://zeiss-campus magnet fsu edu/articles/probes/fpintroduction htmlFrom http://zeiss campus.magnet.fsu.edu/articles/probes/fpintroduction.html

Many Color Variants

Dr R Campbell U of Alberta

See: Fluorescent Proteins By Dr. R. Campbell (Scolarpedia)

Dr. R. Campbell U of Alberta

Focal Adhesion Proteins…Molecular ClutchFocal Adhesion Proteins…Molecular Clutch

CellMembrane5 nm thick

Vicente-Manzanares, M. et al. JCS (2005)

Fluorescence Correlation SpectroscopyFluorescence Correlation Spectroscopy

BeamFocus

4

Intensity FluctuationsLaser Focus Molecular Dynamics

0

2

i>

Intensity Fluctuations~ 1 um3

i-2

<

i(t)=i(t) –<i>Fluorophores excited in focus Number in the

i(t)

f-1 -0.5 0 0.5 1

-4 Fig. 1 Overview of Fluctuation Spectroscopy

i(t) i(t) <i>excited in focus Number in the Focus fluctuates

f

100f 780 920 l 82MH t

LaserLaser--Scanning Microscopy…ImagingScanning Microscopy…Imaging

TiSapph. laserM1

100fs, 780-920nm pulse 82MHz rep-rate

PMT 3 PMT 2 PMT 1Sample

FilterEm. Filters

M4Dichroic i

Filter

Dichroic mirrors

+L1 pinhole+L2M2 M3

mirrorDichroic mirrors

+L1 pinhole+L2M2 M3t=0 t=1 t=2 t=3 t=4

Evanescent Wave Fluorescence ExcitationEvanescent Wave Fluorescence ExcitationTotal Internal Reflection Fluorescence Microscopy (TIRF

Microscopy)

Widefield TIRFZ D th f fi ldZ-Depth of field~ 100 nm at theboundary

http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html

Temporal Image Correlation Spectroscopy (TICS)Temporal Image Correlation Spectroscopy (TICS)

Temporal Autocorrelationof i(x,y,t) = i(x,y,t) - <i>Through Time Series Decay

t=0

Temporal ACF

0.16

0.18

DecaySlowTransport

DynamicsDiffusion Coeff.

t=1

r 11(

0,0,)

0.10

0.12

0.14 &Flow Speeds

Off t

t=2

(s)0 100 200 300 400 500

0.06

0.08 OffsetImmobilePopulation

t=3

t t11 i i

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

t=4

Srivastava and Petersen Methods Cell Sci. 18, 47-54 (1996)t=n

Overview for TalkOverview for Talk

Spatio-Temporal Image Correlation Spectroscopy

k Reciprocal Space Image Correlation p p g

Spectroscopy (kICS)

SpatioSpatio--Temporal Image Correlation SpectroscopyTemporal Image Correlation Spectroscopy

Spatial Correlation as a functionOf Time r11(,,)

t=0

t=1

t=2

tt11 ii

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

t=3

t t Hebert et al. Biophys. J. 88-3601 (2005)t=4

r11(,,)Brownian Flow

t=nDiffusionPeak

FlowPeak Flow

Vector

Vector Maps of Vector Maps of --actinin MEF Cellactinin 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 --actininactinin

-actinin/EGFP in MEF Cell actinin/EGFP in MEF CellTIRF Microscopy

r()

Accelerated40 ti f t

ImmobileFiltered

40 times faster thanReal-time

3.4 μm

τ = 0 s 200 s

Correcting for Immobile or Slowly Moving SpeciesCorrecting for Immobile or Slowly Moving Species

Fourier Filtering (Full Time Stack)Filter pixel stacks in temporal frequency domainWith Heaviside Function before STICS analysisR I bil F t

t=0

MAV Removes Immobile Features t=1

MAV3

t=2

t=3

Moving Average Filter Odd # Framest=4 Moving Average Filter Odd # FramesApplies Immobile Filtering over ShorterTime Window

t=n

Space Time Correlation for Mobile FractionSpace Time Correlation for Mobile Fraction

• Filter Immobile in Frequency Space → r(,,) Mobile

FractionFraction

2D Grey Scale Contour MapS ti l C l ti F ti ( )

• Simulation: 90% immobile

Spatial Correlation Function()

r(,,) vs. 90% immobile, 10% with flow + diffusion in x and y

(,, )For Mobile Fraction

Vector Maps of Vector Maps of --actininactinin

9 μm/min

0 μm/min0 μm/min5 μm

0.7

0.8

min

)r()Inverse relationship b/w

retrograde flow & protrusion speed

0.3

0.4

0.5

0.6ra

de fl

ow (

m/m

r()Ff Fourier Filtered

protrusion speed(similar to actin)

0.0 0.2 0.4 0.6 0.8 1.0 1.20.0

0.1

0.2

Ret

rog

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

()Ff Fourier Filtered

CoCo--Transport of Adhesion Molecules & ActinTransport of Adhesion Molecules & Actin

integrinA F actin

D I

GB illi

talin

5 m1 m/min

ti

actin

GB paxillin

vinculinE J

actin

actin

C HFAK actinC HFAK actin

θVact

Compare everyVector in Ch1 & Ch2For magnitude &

Vgfp

gDirection correlation

Correlated Transport PropertiesCorrelated Transport Properties

1.0

1.2 relative magnitude directional correlation

3T3 Fibroblasts

0 6

0.8

b. u

nits

)

0.4

0.6

Scor

e (a

rb

integrin paxillin FAK talin vinculin a-actinin0.0

0.2

αintegrin paxillin FAK talin vinculin α-actining p

αintegrin paxillin FAK talin vinculin α actinin

Intermediate

~ 70% level

very low magnitude &

low directional correlation

high magnitude &

directional correlation

pax-FAK-talin-vinculin are part of a distinct linkage complex

~ 70% levellow directional correlation directional correlation

Emerging picture of linkageEmerging picture of linkage

actinin

Brown et al. JCS (2006) 119: 5204-5214Faculty of 1000 Selection Jan 2007

-actinin

integrin ECM

actin

g ECM

See Also Ke Hu, et al.Science 5 January 2007: 111-115

Microscopic basis for similar flow fieldsMicroscopic basis for similar flow fields

Always co-localized1 Always co localized

Same speed and direction,not co-localized

2

Same direction,3 different speed

Transient binding/unbinding

3

4and co-transport

4

Mapping the Dance Partners…Mapping the Dance Partners…ChallengeTrying to Resolve the Molecular Dance Partners

4

G i B2

Gaussian Beam Focus

-2

0

1 m3

-1 -0.5 0 0.5 1-4

~ 1 m3

Optical Resolution ~ /2 Macromolecules ~ /50

= Wavelength ~ colour of the light

vs.

g gCross-correlation

Leafs Habsvs.

STICCS: two color crossSTICCS: two color cross--correlationcorrelation

ttii

tyxityxir

21

2112

),,(),,(),,(

tt 21

x y = 1Channel 1 Channel 2

x y = 1Channel 1 Channel 2y = 2

Channel 1 Channel 2

y = 2Channel 1 Channel 2

y = 0Channel 1 Channel 2

y = 0Channel 1 Channel 2

x

t

x

t t

x 2

t

x 2x

y

t

xy

t

No CrossNo Cross--Correlation between integrin & actinCorrelation between integrin & actin

5-integrinEGFP

mRFP-actin CrossCorrelationEGFP

0.01 m/min

0.39 m/min

= 0 14 m/min = 0 18 m/min = 0.14 m/min = 0.18 m/min

Positive ControlPositive ControlActin-GFP, actin-mRFP MEF cell

Cross-l ti

“RFP”A t l ti

GFPA t l ti correlationAutocorrelationAutocorrelation

0.0 m/min

0.55m/min

Strong, consistent GFP, RFP autocorrelations,and cross-correlation

STICCS CHO STICCS CHO 6611‐‐gfpgfp + + PaxillinPaxillin‐‐mcherrymcherry

Unfiltered Fourier filtered

48 μm 48 μm

Video length: 100 seconds

STICCS CHO STICCS CHO 6611--gfpgfp + + PaxillinPaxillin--mcherrymcherry

Auto 16611

Auto 2PaxillinPaxillin

Cross1 x 26611 PaxillinPaxillin 1 x 2

Fourier Filter Frames 51-100 Pixel size 0.21 m

70

80 hist.

60 hist.

60 hist.

0.00 m/min

2.61 m/min

Fourier Filter Frames 51 100

Median 0.81 m/min 0.99 m/min 0.90 m/min

t 2 s

30

40

50

60

70

# of

val

ues

median:0.806av:0.9sd:0.56

20

30

40

50

# of

val

ues

median:0.991av:1.06sd:0.555

20

30

40

50

# of

val

ues

median:0.895av:0.974sd:0.583

0 1 2 3 40

10

20

|v| (m/min)

0 1 2 3 4

0

10

|v| (m/min)

0 1 2 3 4

0

10

|v| (m/min)

Averaged over the Cell

Neuroscience Applications: Growth ConesNeuroscience Applications: Growth Cones

Pathfinding of Chick DRG Neuron Axon Growth Cones

Cytoskeletal Dynamics (both Actin and Microtubules)

3

3.5

4

4.5No NGF50ng/ml NGF

e of

F-a

ctin *

1.5

2

2.5

3

rade

flow

rate

0

0.5

1

Ret

rogr

N=16 eachUtrophinmarker of f actin Disrupt Actin with

blebbistatin<v> = 3.5 m/min+ 50 ng/mL NGFOvernight

Collaboration with Dr. Tadayuki ShimadaProf. Alyson Fournier MNI, McGill

Neuroscience Applications: Growth ConesNeuroscience Applications: Growth Cones

Pathfinding of Chick DRG Neuron Axon Growth Cones

Cytoskeletal Dynamics (both Actin and Microtubules)

EB3 (3 4 images/s)EB3 (3.4 images/s)marker of microtubule tips <v> = 11.4 m/min

We are looking at turning gradients as NGF is applied on id f th th

Collaboration with Dr. Tadayuki ShimadaProf. Alyson Fournier MNI, McGill

one side of the growth cone

Overview for TutorialOverview for Tutorial

Spatio-Temporal Image Correlation Spectroscopy

k Reciprocal Space Image Correlation p p g

Spectroscopy (kICS)

Quantum Dots as Biomolecular LabelsQuantum Dots as Biomolecular LabelsSemiconductor Quantum Dots…Luminescent Nanoparticles

Smaller QD…bl i i

ZnS CapCdSe corebluer emission

Larger QD…reder emissionreder emission

Photostable…Different sizes…

Different Colors, Track proteins

Fluctuation Spectroscopy on QDsFluctuation Spectroscopy on QDs

(CdSe)ZnS (CdSe)ZnS –– Streptavidin (QD605) Streptavidin (QD605) TIRF Illumination CCD TIRF Illumination CCD DetectionDetectionDetection Detection 50ms Integration Time 50ms Integration Time 2000 Frames2000 FramesSee Bachir et al. JAP 99 (2006)Perturbs fluctuation measurements

Single Dot i(t) trace

Nirmal et al. Nature(London) (1996)

Fluctuation Spectroscopy on QDsFluctuation Spectroscopy on QDs

Diffusion only

QD Blinking

QD Blinkingand diffusion

kICS: Reciprocal Space Time CorrelationkICS: Reciprocal Space Time CorrelationImage 2D FT timeImageseries of images correlation

kxx kx

20

40

60

20

40

60

kyy ky

20 40 60 80 100 120

80

100

120

20 40 60 80 100 120

80

100

120t t

Include (t)

Spatial Fluctuations Reciprocal Space2D FFT

Include (t)(1or 0)

See Kolin et al. Biophysical Journal 91 3061-3075 (2006)x y kx ky

Some New Things: kICS kSome New Things: kICS k--space ICSspace ICSk iPoint source

fluorescenceemitters

Imageseries

2D Fouriertransformof images

k-space timecorrelationfunction

20

20

20 40 60 80 100 120

40

60

80

100

120

20 40 60 80 100 120

40

60

80

100

120

constantalinstrumentqt),(*)I( q t),i( rrr

N

io

1

2o

2o

8|| - iexp (t)

2qI t),(i~ krkk

densitynumber cmicroscopi t),(Function SpreadPoint )I(

constant alinstrument q

rr

i 1 82

)t,(i~t),(i~ ),r( * kkk

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

yp),(funtionn correlatio timespace-k

kICS…separtes photophysics & transportkICS…separtes photophysics & transport

200

InterceptPhoto-Physics

SlopeFor a given :

Single -20

0

-5Circularlyaverage

Single

0

20 -15

-10and logtransform

Noise-20 0 20 0 200 400

15

0.4 0.8 1.2-5

5

kICSkICS: Reciprocal Space Time Correlation: Reciprocal Space Time Correlation

kICSkICS: Reciprocal Space Time Correlation: Reciprocal Space Time CorrelationDetermine the slopes for each value of τ, plot them as a function of τ:

Intercept

0 07

-0.06

-0.05

SlopeD independentof o

-0.09

-0.08

-0.07

No non-linearCurve fitting

-0.11

-0.1

g

Do Not need to Calibrate Beam Size!

2 4 6 8

-0.12

0 01

0

2

4

z

2 4 6 8-0.01

00.01

Residuals-1 -0.5 0 0.5 1-4

-2

Measure radii

kICS: Reciprocal Space Time CorrelationkICS: Reciprocal Space Time Correlation

Computer Simulations

Simlulation Parameters128x128 pixels 500 frames Durisic et al. Biophys. J. 128x128 pixels 500 frames0.1 s/frame, 0.1m/pix, 0.4 m PSF, D=0.1 m2/s, mon=2, moff=1.5

p y93-1338 (2007)

QD Labeled CD73 GPI Anchored Protein In CellsQD Labeled CD73 GPI Anchored Protein In Cells

GPI Lipid Anchored Proteinswww.uoguelph.ca/~fsharom/research/gpi.html

IMR-90 Fibroblast CellsCD73 GPI Anchored Proteins Tagged with QDsggImaged at Video Rate (30 fps) (Chris Lagerholm UNC Chapel Hill)

QD Blinking Does Affect Temporal Image QD Blinking Does Affect Temporal Image Correlation…kICS eliminates the problemCorrelation…kICS eliminates the problem

2 1

6 0.00TICS of QD tagged

DkICS = 0.088 ± 0.008 µm2s-1DTICS = 0.109 ± 0.003 µm2s-1

r 11(0

,0,

)

4

[

m2 s-1

]

-0.04

-0.02QD taggedGPI Anchored Receptors

kICS of QD taggedr

2

0 0 0 2 0 4 0 6 0 8

-D

-0.06

QD taggedGPI Anchored Receptors

(s)0 1 2 3

(s)0.0 0.2 0.4 0.6 0.8

Durisic et al Biophys J

Correlation Volume

QD T t

Durisic et al. Biophys. J. 93-1338 (2007)Quantum dot labeledGPI Anchored ReceptorsQD Transport

FluctuationsQD BlinkingFluctuations

GPI Anchored Receptors

kICSkICS separates photoseparates photo--physics & transportphysics & transport

Different particlenumbersnumbers

Different diffusioncoefficientscoefficients

Different blinkingDifferent blinkingor bleaching

Kolin, et al., Biophys. J. 2006 91: 3061-3075.

kICS can measure Changes in BlinkingkICS can measure Changes in Blinking

Th t C l ti F ti

1

Theta Correlation Function

0.6

0.8

(t+)

GFP-adhesion proteinCHO ll

0.4

(t)

( CHO cell

GFP mono-exponential0.2 bleaching

50100

50100 t (s) (s)

Kolin, et al., Biophys. J. 2006 91: 3061-3075.

QD labelling of T cell receptorsQD labelling of T cell receptors

Quantum dot

Collaboration with

•Michael Edidin JHU Quantum dot

Streptavidin• Jon Schneck JHU

MHC monomerwith biotin

T cell receptorCD8 co-receptor

T cell receptor

Drawing by David S. Goodsell

T cell membrane

Qualitative DifferencesQualitative Differences

• Change in blinking of QDs?• Cell size• Change in distribution of QDs?

10

20

30

• Change in distribution of QDs?• Can these be quantified?

5

10

40

50

60

7015

20

25

30

35 20 40 60 80 100

70

80

90 5 µm

Activated cell – Day 4TCRs are more clustered

10 20 30 40 50

40

45

50

55

5 µm

Naïve cell Day 0Epi-fluorescence captured on an EM-CCDCame aCameraResults are for 300-500 image time series averaged over many cells (~20 cells/day)

kICS detects blinking changes in T CellskICS detects blinking changes in T Cells1 01.0 Activated (Day 4)

Theta Ratio = ’

0.8

Activated)>

Theta Ratio ~0.9

0.6

Naive

(t)

(t+

NaiveActivated

0 4

<

Theta Ratio ~0.4

NaiveActivated Clustered QD/TCRsBlink Less Frequently

0.4

kICS detects this(simulations support this)

1 10 (s)Yu & Van Orden PRL 2006 & Scheibner et al. Nat. Phys. 2007

ConclusionsConclusions

Spatio-Temporal Image Correlation Spectroscopy

Transport maps of FP labeled proteins in cellsp p p

k Reciprocal Space Image Correlation

Spectroscopy (kICS)

Separates photo-physics and transport fluctuationsp p p y p

Data mining the images (there’s gold in those hills

(fl t ti ))(fluctuations))

AcknowledgementsAcknowledgements

• Michael Edidin JHU• Allan “Rick” Horwitz UVaSTICS Molecular Clutch kICS

• Jon Schneck JHU

• The Schneck lab

• Claire Brown UVa & McGill University

• Ben Hebert UVa

• David Kolin

• David Ronis

• David Kolin

• Tim ToplakJohns Hopkins

U i it • David Ronis

• Nela Durisic

Ch i L h l UNC

• Tim Toplak

• Tadayuki Shimada (MNI)

l ( )

University

• Chris Lagerholm UNC

•Jeremy

• Alyson Fournier (MNI)

Schwartzentruber

Thanks to the Group! Thanks to the Group!

Ben HebertDavid KolinNela Durisic David KolinNela Durisic

Thanks to Prof Rick Horwitz UVa & Dr. Claire Brown McGill

Thanks to the Group! Thanks to the Group!

Tim Toplak

& Jeremy Schwartzentruber, Dominique GuilletSebastien Cote, Benoit Vallaincourt

Correlated Transport Depends on [ECM]Correlated Transport Depends on [ECM]

1.0Directional Correlation

B

P illiLow = 2 g/ml

EGFP Protein mRFP-Actin

2 g/ml Directional

arb.

uni

ts)

0.6

0.8

Talin

PaxillinLow 2 g/mlHigh = 5 g/mlFibronectin

Talin

5 g/ml

Correlation

Sco

re (a

0.2

0.45 g/ml

FibronectinTalin

0.0

Low High HighLow5 g/ml

Fibronectin [FN]

Paxillin

Quantum dot blinkingQuantum dot blinking

Intensity1

ton

toffy

time

on off 0off

On and off time distributions have “power-law” decays• Fractal• Occur on all timescales• Occur on all timescales• ton and toff have power law distribution

mon depends on:

Log(Probability) slope:mon

mon depends on:• Core• Shell• Surface functionalization

E it ti i t it

Log(ton)

• Excitation intensity• Ligands in solution (e.g., pH)• Manufacturer/Batch of QDs

“ l ” bli k

Three blinking simulationsThree blinking simulations

Intensity

on off

“slow” blinker

0

1

time

on off

“fast” blinker

1Intensity

on off0

1

timetetramer “slow” blinking4

Intensity

0time

0

0

kICS can measure blinking within clusterskICS can measure blinking within clusters

y = -0.1119x - 0.0432R2 = 0.9931

-0.2

-0.10 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

) Computer

0 5

-0.4

-0.3

t)the

ta(t+

tau)

>

slow blinkingtetramers

f t bli ki slow blinking

ComputerSimulations

PSF I t t d

-0.7

-0.6

-0.5

ln(<

thet

a(t fast blinking

monomersslow blinkingmonomers

PSF Integrated

0

2

4

y = -0.2631x - 0.507R2 = 0.9873

-0.9

-0.8

ln(tau)

-1 -0.5 0 0.5 1-4

-2

ln(tau)

Dot-taggedproteins

aggregategg g

Degree of aggregationDegree of aggregationAdd T C ll G th F t D 6 & 10 ti ti hAdd T Cell Growth Factor Days 6 & 10…activation echo

1000

ion

100Aggr

egat

Deg

ree

of

10

D

Naive 1 2 3 4 5 6 7 8 9 10 11 12

Days after activation

Diffusion coefficientDiffusion coefficient

0.01

m2 /s

)oe

ffici

ent (

iffus

ion

co

0 00

Di

Naive 1 2 3 4 5 6 7 8 9 10 11 120.00

Days after activation

QD clusters blink differently than single QDsQD clusters blink differently than single QDs

Single QDSingle QD

QDCluster

As well Scheibner et al. Nature Physics 3:106–110, 2007