cours biophotonique 3 2012 - École polytechnique
TRANSCRIPT
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
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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
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600
800
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1000
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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().
D. Marguet et al., EMBO J. 25, 3446 (2006).
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
E. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007)
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
E. Haustein & P. Schwille, Annu. Rev. Biophys. Biomol. Struct. 36, 151 (2007)
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
D. Marguet et al., EMBO J. 25, 3446 (2006).
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.