cours biophotonique 3 2012 - École polytechnique

Fluorescence techniques Single‐molecule optical microscopy

• Principle and instrumentation• Localization precision• Applications to biomolecule conformational changes• Applications to biomolecule diffusion and cell dynamics

Fluorescence Resonant Energy Transfer (FRET)• Förster model• Applications

Fluorescence lifetime imaging (FLIM)• Time‐domain and frequency‐domain implementations• Applications

Fluorescence recovery after photobleaching (FRAP) Fluorescence correlation (FCS)

• Principle• Applications

Antigoni [email protected], 01.69.33.50.04

COURSE 3: Fluorescence microscopy (Part II)

« Biophotonics » course

Single‐molecule signaturesDiffraction‐limited emission spotSignal intensity

0 2 4 6 8 10 12

0

200

400

600

800

Coun

t num

ber

Time (s)

1000

0 5 10 15 20 25 30

0

100

200

D. Casanova et al., J. Am. Chem. Soc. 129, 12592 (2007)

For organic fluorophores: Photobleaching steps

For Quantum Dots (QDs): Blinking

X. Brokman et al, Phys. Rev. Lett.90,120601(2003)

For single‐photon sources: Photon anti‐bunching

X. Brokman et al, Phys. Rev. Lett.90,120601(2003)

CdSe/ZnS QDs, lifetime 20 ns

D. Casanova, S. Türkcan, LOB

Hanbury‐Brown‐Twisssetup

Visualization of single ATP turnovers in myosin using TIRFM

T. Yanagida group, Osaka UniversityT. Funatsu et al., Nature 374, 555 (1995).

M. Tokunaga et al., Biochem. Biophys. Res. Comm. 235, 47 (1997).

BDTC‐S1: headportion (S1) of myosin whichcontains both the actin‐binding site and the catalytic(ATP hydrolysis)site modified witha biotin‐bindingpeptide (BDTC)

Fluorescent ATP

When Cy3‐ATP (or Cy3‐ADP) is bound to the myosin head, it appearsas a bright fluorescent spot.When it is free, itundergoes rapidBrownian motion awayfor the surface and is not visible as a discrete spot.

ATP synthesis: Oxidative phosphorylation

Mitochondrial matrix

Why oxidative phosphorylation ?Phosphorylation because a phosphate group is added to ADPOxidative because oxygen is used in the process

Functioning of ATP synthase

Protein Data Bankhttp://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb72_1.html

John Walker, Medical Research Center, Mitochondrial Biology Unit, Cambridge, UKhttp://www.mrc‐mbu.cam.ac.uk/research/atp‐synthase

Red: open stateOrange : loose statePink: tight state

membrane

F1 portion:3 and 3 subunitsRotating subunitinduces conformationalchanges of and subunits

http://rsb.info.nih.gov/NeuoChem/biomach/ATPsyn.html

Modified from

When dissociated from a proton gradient, ATP synthase acts as an ATPase(counterclockwise rotation).

Rotation of the ‐subunit of F1‐ATPase

K. Kinosita Jr group, YokohamaH. Noji et al., Nature 386, 299(1997)

In the presence of 20 mM ATPNo rotation without ATP

Single‐moleculeMultiple‐fluorophore observation

Rotation of the ‐subunit of F1‐ATPase

K. Kinosita Jr group, Yokohama

P. Selvin’s group, University of IllinoisYildiz et al., Science 300, 2061 (2003).

Myosin V motion on actin filamentsIn vitro

Myosin V motion on actin filaments

Yildiz et al., Science 300, 2061 (2003).

Cy3 or bisiodoacetamidorhodaminelabeling

Oxygen leads to fluorophoreoxidation and photobleaching‐> Oxygen scavengingconditions:Glucose + glucose oxidase + catalase

Obtained 10000 photons in 0.5 s‐> localization precision: <1.5 nm !!Immobile myosin withoutATPLow ATP concentration (300 nM ATP) led to decreased stepping rate and discernable steps

Alternating 52‐23 nm steps are compatiblewith a dye located at 6.5‐7 nm from the midpoint in the direction of motion‐> myosin V walks hand over hand

Measuring distances: Beyond the Rayleigh criterionFor two identical, self‐luminous, in‐focus point sources emitting unpolarized, incoherent light separated by a distance d, the fundamental resolution measure(FREM) depends on the number of photons

na: numerical aperture of the objective lens0: photon detection rate per point source [t0, t]: acquisition time intervalJn: nth order Bessel function of the first kind

an2

After adding pixellisation noise, background fluorescence (Poisson) noise, and detector readout noise (Gaussian), the practicalresolution measure (PREM) is obtained. S. Ram, E. S. Ward, and R. J. Ober, PNAS 103, 4457 (2006)

d=30 nm40 nm

50 nm

with noisewithout

Resolution as a function of the distance d

For an experiment wherethe Rayleigh criteriongives: 290 nm

2500 photonsPoisson noise: 80 photons/s

Ultrahigh‐resolutionmulticolor colocalizationof single fluorescent probes

S. Weiss groupT. Lacoste et al., PNAS 97, 9461 (2000)

200 nm

QDs emitting at540 nm (green)and 620 nm (red)

DC1, DC2 : dichroic mirrorsPH: pinhole

Myosin V walks hand over hand one color per head

D. M. Warshaw, Biophys. J. 88, L30 (2005)

QD labeling

Localization precision: ~6 nm

Green images shifted verticallyby 12 pixels (660 nm) for clarity

Subunit counting in membrane‐bound proteins

Ulbrich & Isacoff, Nature Meth. 4, 319 (2007)

GFP labeling, 1:1 stochiometryCyclic nucleotidegated channel

LOB & PMC, Ecole PolytechniqueD. Casanova et al., J. Am. Chem. Soc. 129, 12592 (2007)

NP channel(617 nm)

Alexa channel(519 nm)

Counting the number of proteins bound to single nanoparticles

0 12

3

45

>5

0 1

2

34

5

>5

1

3 Alexa/NP2

4

5

2

1

Alexa/proteinAlexa/NP

proteins/NP

0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0 6 0 0 00

5

1 0

1 5

2 0

2 5

Num

ber o

f em

issi

on s

pots

Initial count number

AlexaY1‐xEuxVO4

siloxanepolymer

luminescent nanoparticle

proteinAlexa

Alexa

organicfluorophore

0 2 4 6 8 10 12

0

200

400

600

800

Coun

t num

ber

Time (s)

1000

0 5 10 15 20 25 30

0

100

200

Observing protein synthesis one molecule at a time

Sunney Xie group, Harvard UniversityJ. Yu et al., Science 311, 1600 (2006).

Fusion protein of Venus (a YFP variant) and a membrane protein (Tsr)

Single‐stepphotobleaching

Principle: observation by immobilization

BacteriumE. Coli

Observing protein synthesis one molecule at a time

Sunney Xie group, Harvard UniversityJ. Yu et al., Science 311, 1600 (2006).

Every 3 min observe produced proteins, then photobleach the Venus molecules.‐> proteins are produced in random bursts, a variable number is synthesized each time

One mRNA copy gives rise to each burst; lifetime 1.5 minAverage number of proteins produced per burst: 4.2

Dual‐color detection and dual‐polarization detection

T. Schmidt group, Leiden Univ., The NetherlandsL. Cognet et al, Appl. Phys. Lett. 77, 4052 (2000)

Measure simultaneously 2 different fluorophoresand their 2D orientations (‐> rotational dynamics)

Single‐molecule observations to study diffusionExample: Confined motion of a membrane receptor

S. Türkcan, LOB, Ecole Polytechnique

Single‐molecule observations to study diffusionAnalysis using the mean square displacement (MSD)

M. J. Saxton, K. Jacobson, Annu. Rev. Biophys. Biomol. Struct. 26, 373 (1997).

N framest: time interval between images

Two‐dimensional mean square displacement (MSD)

Analysis using the mean square displacement (MSD)

A. Kusumi et al, Biophys. J. 65, 2021 (1993).

Case c: free Brownian diffusion inside an infinitely high square well potential

M. J. Saxton, K. Jacobson, Annu. Rev. Biophys. Biomol. Struct. 26, 373 (1997).

Brownianmotion

Directedmotion

Confinedmotion

Case a: free Brownian diffusion

Case a: directed diffusion with a constant drift velocity v

In all cases, the initial slope gives the diffusion coefficient D.

Time interval

M. Dahan et al., Science 302, 442 (2003).

Diffusion of glycine receptors revealed by single‐QD tracking

Free diffusion Confined diffusion

Red : synaptic boutonsGreen : QDs – glycine receptors

Extrasynaptic motion Motion at the synapse

Diffusion of GABA receptors in nerve growth conesrevealed by single‐QD tracking

C. Bouzigues & M. Dahan, Biophys. J. 92, 654 (2007)C. Bouzigues et al., Proc. Natl. Acad. Sci USA 104, 11251 (2007)

Alternating directed and Brownian motion

White‐light transmissionImaging of QDs labeling

GABA receptors

C. Bouzigues

Use a speed correlation index to determine the portions of the trajectory corresponding to directed motion.

Red: QDsGreen: MicrotubulesBlue: Nucleus

B. Lounis, L. Cognet & D. Choquet groups, Univ. BordeauxLasne et al., Biophys. J. 91, 4598 (2006).

Photothermal tracking of 5‐nm gold nanoparticles

Track 5‐nm gold nanoparticles withphotothermal heterodyne imagingAdvantage:Very small particles are detectableNo blinkingNo photobleachingDisadvantage:It is not a wide‐field techniqueSolution:Follow a single nanoparticle using a triangulation procedure‐ Locate a NP‐Measure 3 data points around this position at the apices of an equilateral triangle‐> Determine (xt, yt, St)‐Measure 3 new data points after recenteringthe equilateral triangle‐> Determine (xt+t, yt+t, St+t)

Analyzing trajectories using the inference approach

Institut Pasteur & LOB, Ecole PolytechniqueJ.‐B. Masson et al., Phys. Rev. Lett. 2009

Equation of Motion

)()( 2 tDxVvdtdvm xx

x

Thermal noisePotentialFriction

DrVdtdr 2)(

This means that the system relaxes quickly:

sskg

kgm 166

22

10/10

10

PDPFPt )(1

The associated Fokker‐Planck equation is:

0ttt

For constant D and F, the solution is:

Let us consider free Brownian motion + a force

tD

tDtFrr

trtrP

44

)/(exp,|,

20

00

00111122

11

,|,,|,...,|,,

trtrPtrtrPtrtrPFDTP NNNN

For a trajectory T starting atand ending at:

00 , trNN tr ,

Analyzing trajectories using the inference approach‐> When you know F and D, you can calculate the probability to go from the space‐time point (r0,t0) to the space‐time point (r,t) We are in the inverse situation: We know that the molecule went from the space‐time point (r0,t0) to the space‐time point (r,t) and want to calculate the probabilitythat this happened for a certain value of parameters F and D.

Bayes theorem:

)()()|()|( 0

TPQPQTPTQP

Posteriorprobabilityof having the parameter values Q given the observation of the trajectory T

Probability of observing the trajectory T given the parameter values Q

Prior probability = constant

NormalizationConstant

In our case:T: trajectoryQ: parameters F, D

J.‐B. Masson et al., Phys. Rev. Lett. 2009

Analyzing trajectories using the inference approach

J.‐B. Masson et al., Phys. Rev. Lett. 2009

Trajectory: confined motion of a membrane receptor Map of the forces felt by the receptor

inference

5.5 5.6 5.7 5.8 5.9 6.0

5.7

5.8

5.9

6.0

6.1

Posi

tion

[µm

]

Position [µm]

K. Jacobson, E. Sheets & R. Simson, Science 268, 1441 (1995)

Different types of membrane compartmentation

« The inner life of a cell », University of Harvard

Cytoskeleton boundaries Lipid rafts

Variants for single‐molecule spectroscopyPrism TIRF Objective TIRFWide‐field

Narrowfield

N. G. Walter et al., Nature Methods 5, 475 (2008).

Scanning near‐fieldoptical microscopy

SNOM

Comparison epi‐fluor.TIRF

Confinement in microfluidic channels

Confinement in capillaries

Width smaller than the diffraction limit15‐100 nm

3D tracking using a cylindical lens

H. P. Kao and A. S. Verkman, Biophys. J. 67, 1291 (1994).L. Holtzer, T. Meckel, and T. Schmidt, Appl. Phys. Lett. 90, 053902 (2007)

A cylindical lens introduces astigmatism.

Withoutcylindrical lens, no differencebetween+z and ‐z

zr: focal depth0: diffraction‐limited FWHM for a point source at focus

3D tracking using a cylindical lens

L. Holtzer, et al, Appl. Phys. Lett. 90, 053902 (2007)

Without cylindrical lens, the z‐position can bedetermined from the FWHM of the intensitydistribution,

With cylindrical lens, the z‐position can be determined from the width, r, and the ellipticity, , of the intensity distribution:

With cylindrical lens:• Improved axial accuracy• Lateral accuracyonly slightly worse

is the axial astigmatism of the lens

3D tracking of QDs inside live cells using a cylindical lens

L. Holtzer, T. Meckel, and T. Schmidt, Appl. Phys. Lett. 90, 053902 (2007)

exposure time: 6 msx, y localization precision: 43 nmz localization precision: 130 nm

Cylindrical lensf=10 m

Fluorescence techniquesFörster resonant energy transfer (FRET)

Förster resonant energy transfer (FRET)

Donor Acceptor

donor excitation donor emission

energy transfer

acceptor emission

In the presence of resonant energy transfer: decrease of donor emission increase of acceptor emission decrease of donor lifetime

Extensively used in biology to observe distance changes (2‐10 nm):‐ Interaction between biomolecules‐ Conformational changes

Energy transfer depends on the 6th power of donor‐acceptor distance‐> spectroscopic ruler

T. Förster, Annalen der Physik 2, 55 (1948)R. M. Clegg, Curr. Op. Biotech. 6, 103 (1995)

P. R. Selvin, Methods in Enzymology, Vol. 124, Academic Press (1995), p. 300

Non‐radiative energytransfer from the excited donor to the acceptor molecule via dipole‐dipole coupling

Prerequisites:• Overlap between donor emission and acceptor absorption spectra

ikrDDDD e

rik

rpprrrpr

rkrE

230000

2

0

1)(3)(4

1)(

Point dipole approximationA harmonically oscillating electric dipole pD produces an electric field:

ikrDDD e

rpprrE

300

0

)(34

1

In the near field (r<<):

Coupling with another point dipole :

ikRDADADA e

RppprprEpV

300

0

.))((34

1.

Ap

Fermi’s golden rule gives the transition rate from the donor excited state to the acceptor excited state:

Förster resonant energy transfer (FRET)Nonradiative induced dipole – induced dipole interaction

R

0rDp Ap

62 12

RfVik fT

ADi **ADf

rrr 0

: unit vector joining the two dipoles

Initial state

0r

R: distance between the two dipoles

fFinal state

Density of final states

Interactionenergy

D: donorA: acceptor

Förster resonant energy transfer (FRET)

Jablonski energylevel diagram

E: efficiency of energy transfer (or FRET efficiency), defined as the probability for the excited donor to return to its ground state by energy transfer to the acceptor

6

01

1

RR

E

Energy transfer rate and efficiency

J: normalized spectral overlap of the donor emission (fD) and acceptor absorption (A) in units of M‐1 cm‐1

2: depends on relative dipole orientation qD: donor quantum yield, n: refraction index

60

RRkk DT

DT

Tkk

kE

:Tk:Dk total rate of all other radiative and non‐radiative donor decay processes

rate of energy transfer to the acceptor

DD k

1

R0: the distance R for which the energy transferrate is equal to the donor excited‐state decay rate

R0 ~ 5‐7 nmDistance measurements: ~2‐10 nm

T. Förster, Annalen der Physik 2, 55 (1948)P. R. Selvin, Annu. Rev. Biophys. Biomol. Struct. 31, 275 (2002)

P. R. Selvin, Methods in Enzymology, Vol. 124, Academic Press (1995), p. 300

Measurements of energy transfer efficiency

D

D

II

E A1

D

DAE

1excited‐state lifetime of the donor in the absence of the acceptorexcited‐state lifetime of the donor in the presence of the acceptor

:D:

AD

:ADI

donor emission intensity in the absence of the acceptordonor emission intensity in the presence of the acceptor

:DI

Intensity measurements: beware of pitfalls‐> In ensemble measurements, intensities must be normalized for concentration.

The lifetime approach is concentration independent.

Alternatively:

DDAA

AA

qIqIqI

EAD

D

///

:DAI FRET‐induced acceptor emission intensity

Beware of direct acceptor excitation

P. R. Selvin, Annu. Rev. Biophys. Biomol. Struct. 31, 275 (2002)

Donor (acceptor) quantum yield:)( ADq

Rewording the FRET efficiency definition: E is the donor fraction de‐excited via energytransfer to the acceptor

FRET donor‐acceptor pairsA large variety of FRET pairs exists:• Dye‐>dye (ex. Cy3‐> Cy5)• Fluorescent protein‐> fluorescent protein• Nanoparticle ‐> dye• ….

Quantum dot ‐> dye

H. Mattoussi group, Naval Res. LabA. R. Clapp et al.,

J. Am. Chem. Soc. 126, 301 (2004)

Energy transfer from a QD donor to multiple organic acceptors

For 1 donor interacting withn acceptors:

H. Mattoussi group, Naval Res. LabA. R. Clapp et al., J. Am. Chem. Soc. 126, 301 (2004)

QD‐peptide‐dye conjugates to monitor proteolytic activity

I. L. Medintz et al., Nature Materials 5, 582 (2006)

Thrombin cleaves the peptide attaching the QD to the acceptordye.The thrombin inhibitor inhibitsthe thrombin enzyme activity.

QD‐dye conjugates as energy‐transfer‐based pH sensors

Bawendi group, MITM. Bawendi and coll., JACS 128, 13320 (2006)

pH 6.0

pH 10.0

Spectral shift of the acceptor dye upon protonation at low pH‐> better overlap, higher FRET efficiency

Detection of ERK receptor phosphorylation using FRET

Harvey et al, Proc. Natl. Acad. Sci. USA 105, 19264 (2008).

Stimulation by epidermalgrowth factor (EGF) inducesReceptor phosphorylation Donor lifetime changes due to FRET

Single‐pair FRET (sp‐FRET)Confocal microscope

P. G. Schultz and S. Weiss groupsA. A. Deniz et al., Proc. Natl. Acad. Sci. USA 96, 3670, (1999)

donoronly

• Results in agreement with the dye distance• Detection of different subpopulations• Measurement of enzymatic cleavage activity

Cleavage with a restriction endonuclease

B. Schuler, E. A. Lipman, W. A. Eaton, Nature 419, 743 (2002)

Folded vs. unfolded proteins using FRETfoldedunfoldeddonor

only

Limit on reconfiguration time imposes limit on free‐energy barrier

Addition of a denaturant

Exploiting long lifetime donors : the case of rare‐earth ions

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

0

1 0

2 0

3 0

4 0

5 0

Inte

nsity

(arb

. uni

ts)

T im e (µ s )

Cy5

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

0 ,0 1

0 ,1

1

Nor

mal

ized

inte

nsity

T im e (µ s )

NP

NP-Cy5NP

NP-Cy5

DonorNP

AcceptorCy5

donor excitation

donor emission

energy transfer

acceptor emission

0 20 0 4 00 6 00 8 00-2

0

2

4

6

8

10

Inte

nsity

(arb

. uni

t)

T im e in µ s

NP‐Cy5 ex: 466 nm

Cy5

NP‐Cy5 ex: 457 nm

Excitation chopper closing

Detection chopper opening

Acceptor (Cy5) emission

Donor (Eu‐doped nanoparticle) emission

D. Casanova, D. Giaume, T. Gacoin, J.‐P. Boilot, A. Alexandrou, J. Phys. Chem. B. 110, 19264 (2006)

P. Selvin, Annu. Rev. Biophys. Biomol. Struct. 31, 275 (2002)

Fluorescence techniquesFluorescence lifetime imaging

Fluorescence lifetime imaging (FLIM)

nradrad

rad

kkkq

nradrad kk

1

The fluorescence quantum yield q is determined by the radiative and nonradiative transition rates krad and knrad and issensitive to the local environment.

It is easier to measure the lifetime .

J. R. Lakowicz, Principles of Fluorescence Spectroscopy, (Plenum, New York, 1983).

In biological samples, fluorophore lifetime can depend on [Ca2+], [pO2], pH

Example: low pH in cancer tissues

krad knrad

2D‐Fluorescence lifetime imaging (2D‐FLIM)Detection using a gated image intensifier

P. French website Dowling et al, Opt. Comm. 135, 27 (1997).

Time‐domain lifetime measurements

Acceleration voltageCan be switched on and off‐> gating

photons

photocathode

Electronacceleration

Phosphor screen

photons

CCD

t

V

75 ps‐2 ns

• Wide‐field technique• Only the photons arriving during the acceleration gate are detected.

Time‐domain lifetime measurementsTime‐correlated single‐photon countingPrinciple:

• Periodic light signal (e. g. excited by a pulsed laser)• Fast electronics (TAC:Time to amplitude converter) to detect the arrival time of individual photons• Reconstruction of the waveform from the individualarrival time measurements.Requirement: Not more than one photon in one period

Becker & Hickl GmbH website

• Point‐by‐point detection (single detector).• No photons are lost. All photons are detected.‐> optimum signal‐to‐noise ratio for a givennumber of detected photonsBUT: the signal has to be reduced to remain in the single‐photon counting regime.

Combination with scanning: confocalor non‐linear microscopyAlso used for fluorescence correlation

Detectors:Avalanche photodiodes (APD)Photomultipliers

Pulsedexcitation source

Time to amplitude converter(TAC)

stop

start

Data display

Excitation

Sample

Fluorescence

ADCDetector

Gated image intensifiers vs. time‐correlated single‐photon counting

Highest accuracy per photonRequires scanningBest for FLIM with a confocal microscope

Highest accuracy per second2D signal output Best for applications like endoscopy

Gated image intensifiers Time‐correlated single‐photon counting

Time‐domain lifetime measurementsStreak camera

Advantages: High temporal resolution (~1 ps), adequate for short lifetimes (ns or sub‐ns)Disadvantages: ExpensiveLow lifetime dynamic range

slit

photo‐cathode

‐

+

anodeMicrochannelplate (MCP)

phosphorscreen

intensity

time

e‐photons

t

V

‐ +

CCD

MCP

Frequency‐domain lifetime measurements

J. R. Lakowicz, Principles of fluorescence spectroscopy, 3rd edition, Springer, 2006

Excitation intensity:

Emission intensity:

‐> Phase shift ‐> Demodulation factor:

For a monoexponential decay:

For multiexponential decays, measurements at multiple frequencies are necessary.

A. Deniset‐Besseau, thesis Université d’Orsay, 2008

Inexpensive technique, but complicated analysis for non monoexponential decays.

Adequate for long lifetimes (µs‐ms).

FLIM variants

FLIM (2D‐gated detection) + optical sectioning

P. French + T. Wilson groupsCole et al, Opt. Lett. 25, 1361 (2000).

FLIM

FLIM

FLIM+structured illumination

FLIM+structured illumination

z=z0

z=z0+315 µm

FLIM variants

P. French + T. Wilson groupsSiegel et al, Opt. Lett. 26, 1338 (2001).

FLIM (2D‐gated detection) + spectral measurements + optical sectioning

FLIM to study ordered lipid domainsdye

di‐4‐ANEPPDHQ

L. Jin et al., Biophys. J. 89, L04 (2005).

Spectral blueshift in the ordered lipid phase

Lifetime increase in the ordered lipid phase

P. French + A. Magee groupsD. M. Owen, Biophys. J. 90, L80 (2006).

LUV: Large Unilamellar VesiclesDOPC: 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholineDPPC: 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholinePSM: n‐palmitoyl‐sphingomyelin

Orderedlipid phase

Disorderedlipid phase

FLIM to study ordered lipid domains

P. French + A. Magee groupsD. M. Owen, Biophys. J. 90, L80 (2006).

Enhanced contrast with lifetime imaging

Effect of temperature and cholesterol depletion

Fluorescence techniquesFluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP)

D. Marguet et al., EMBO J. 25, 3446 (2006).

Ensemble method Difficult to differentiate between different subpopulations

Fluorescence techniquesFluorescence correlation spectroscopy

E. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007)

Fluorescence correlation spectroscopy (FCS)Developed in the 1970s as a miniaturization of dynamic light scattering

Principle: Excited molecules in the focal volume give rise to a fluorescence signal.The fluorescence signal fluctuates in time ‐> F(t).These fluctuations can be quantified by calculating the normalized autocorrelationfunction G().


Confocal setup

Fluorescence correlation spectroscopy (FCS)

The fluorescence fluctuationsmay result from: a change in the number of fluorophores in the observation volume due to diffusion a change in fluorescence properties of the molecule as a consequence of

• a chemical reaction • a conformational fluctuation.

Optimize signal‐to‐noise ratio:Here, the fluctuations are the signal.The relative fluorescence fluctuations increase for decreasing numbers of emittingmolecules‐> minimize the average number of emitting molecules/particles in the focal volume (0.1 to 1000) ‐> concentrations 10‐10‐10‐6 M for a focal volume of ~1fL‐> minimize the focal volume, reduce the emitter concentration

1111)(

)()0(

2

2

2

NNN

tF

tFG

Amplitude at =0 gives the number of molecules in the focal volume Veff

Correction termdue to the fact thatthe focal volume isnot spherical.

3D brownian diffusionFor a depth/diameter (1/e2) ratio S of the gaussian 3D excitation volume:

Fluorescence correlation spectroscopySignal due to diffusion

lateral diffusion time D: Time during which the moleculestays in the focal volume

J. Widengren, R. Rigler, Cell Mol. Biol. 44, 857 (1998) E. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007)

Dr

D 4

20

2D brownian diffusion (e.g. in a membrane)

1

+ 1

r0: lateral 1/e2diameter

Fluorescence correlation spectroscopy: different diffusion models

Brownian diffusion + directed flow with velocity v


1/S2

Directed flow

FCS: fluorophore photophysics complicates the interpretation

If the photophysics does not influence the diffusion dynamics:

BUT: Gphotoph() is not always known

)()()( diffphotoph GGG

More generally:For a chemical reaction or conformational change of the diffusing species witha characteristic time R:

A: fraction of the molecules that undergoes the change

FCS applications‐ Determine concentrations, esp. concentration changes (S. Charier et al, J. Am. Chem. Soc. 127, 15491 (2005)) ‐ Determine the diffusion coefficient‐ Determine the particle size

Stokes‐Einstein equation

‐ Detect binding processes in vitro and in vivo: mass change leads to a change in diffusion coefficient

for a spherical particle in solution : , mmolecular mass‐ Detect events on very short time scales: rotational diffusion, isomerization, …

V: viscosityRh: hydrodynamic radius (size+solvation shell)

3 mD


Advantages: can measure much faster dynamic processesDrawbacks: accurate calibration of the focal volume is important

requires fitting of the data with a model

Comparison to single‐molecule tracking

FCS applications: measure diffusion

Giant Unilamellar VesicleGreen: dye in the liquid‐ordered phase (Alexa488‐labeled cholera toxin B‐subunit)Red: dye in the liquid‐disordered phase (dye diI)Slower diffusion in the liquid‐ordered phase.

P. Schwille groupK. Bacia et al, Biophys. J. 87, 1034 (2004)

2D and 3D images with scanning

FCS applications: detect conformational changes

C. Frieden groupK. Chattopadhyay et al, Biophys. J. 88, 1413 (2005)

IFABP (intestinal fatty acid binding protein, 15 kDa)

Guanidine hydrochlorideinduces unfolding

Beware of modifications of the refractive index mismatch

Change of diffusion coefficient upon unfolding

Dual‐color Fluorescence Cross‐Correlation (FCCS)

P. Schwille et al., Biophys. J. 72, 1878 (1997)E. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007)

If the excitation volume Veff isidentical for the two excitation wavelengths andthere is negligible overlap betweenemission and absorption spectra :

Mij is the motion‐related part of the correlation function

Cross‐correlation between the signals Fi and Fj:

The amplitude of Gx() is proportional to the concentration of doubly labeled molecules.

Red channel autocorrel.Green channel autocorrel.

Cross‐correlation

P. Schwille et al., Biophys. J. 72, 1878 (1997)E. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007)

Dual‐color Fluorescence Cross‐Correlation (FCCS)

+

Reaction kinetics

‐> k=8x105 M‐1s‐1

FCS variants

E. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007)Z. Petrasek & P. Schwille, Scanning FCS, in Single molecules and Nanotechnology, R. Rigler, H. Vogel (eds.), Springer, 2008

Scanning FCS

Dual‐spot FCS

Image correlation FCS

2)(

)()()(

pF

ppFpFpG

More generally: Where p can be any

combination of x, y, z, t

‐> flow measurements in microstructured channelsUse EM‐CCD for multiple spot detection

Spatiotemporal image correlation FCS

Comparison FRAP, FCS, SPT


Time resolution: ~70 ns (APD)

Bibliography (course 3)J. R. Lakowicz, Principles of fluorescence spectroscopy, 3rd edition, Springer, 2006R. Rigler, H. Vogel (eds.), Single molecules and Nanotechnology, Springer, 2008P. Selvin, T. Ha (eds.), Single‐Molecule Techniques: A Laboratory Manual , CSH Lab. Press, 2008

Cited publications, in particular:P. R. Selvin, Methods in Enzymology, Vol. 124, Academic Press (1995), p. 300R. M. Clegg, Curr. Op. Biotech. 6, 103 (1995) FRETP. R. Selvin, Annu. Rev. Biophys. Biomol. Struct. 31, 275 (2002) FRETC. Joo et al., Annu. Rev. Biochem. 77, 51 (2008) single‐molecule and FRETE. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007) fluorescence corr.D. Marguet et al., EMBO J. 25, 3446 (2006) fluorescence correlation

Biophotonics seminarsMonday morning February 6: 10h45‐12h15 Ana‐Maria Pena, L’Oréal

Exam: Wednesday February 15 14h‐17hWritten exam (1h30 on the 3 ½ courses by A. Alexandrou, 1h30 on the rest of the courses)

All documents authorized.

Bibliography projects upon request.

cours biophotonique 3 2012 - École polytechnique

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