critique (everyone) monitoring dynamic protein interactions with photoquenching
DESCRIPTION
Suggested presentation. “Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumor” Jain et al, Nature Medicine, vol 10 203-7 (2004). Critique (everyone) Monitoring dynamic protein interactions with photoquenching - PowerPoint PPT PresentationTRANSCRIPT
Critique (everyone)Monitoring dynamic protein interactions with photoquenching FRET. Nature Methods, vol 3, 519-24 (2006)
“Two-photon fluorescence correlation microscopy reveals the two-phase nature of transport in tumor” Jain et al, Nature Medicine, vol 10 203-7 (2004)
Suggested presentation
1. Campagnola, P.J., et al., High resolution non-linear optical microscopy of living cells by second harmonic generation. Biophys. J., 1999. 77: p. 3341-3349.
2. Campagnola, P.J., et al., 3-Dimesional High-Resolution Second Harmonic Generation Imaging of Endogenous Structural Proteins in Biological Tissues. Biophys. J., 2002. 82: p. 493-508.
3. Moreaux, L., O. Sandre, and J. Mertz, Membrane imaging by second-harmonic generation microscopy. J. Opt. Soc. Am. B, 2000. 17: p. 1685-1694.
Assigned Reading for Next Week
Outline:
1) Fluorescence Lifetime Imaging (FLIM)
2) Fluorescence Resonance Energy Transfer (FRET)
3) FRET/FLIM
4) FCS
Fluorescence Lifetime motivation
1) Sensitive to environment: pH, ions, potentialSNARF, Calcium Green, CameleonsPerform in vitro calibrations
1) Results Not sensitive to bleaching artifacts
2) Not sensitive to uneven staining (unless self-quenched)
3) Not sensitive to alignment (intensity artifacts)
iscf
f
kk
k
nonradiscf
f
kkk
k
iscf kk 1
Fluorescence Quantum Yield φ: important for dyesRatio of emitted to absorbed photons
Measured lifetime is sum ofRates of natural lifetime and non radiative decay paths
(k is rate,(k is rate,Inverse of time)Inverse of time)
Quantum Yield:Quantum Yield:
fk 1
0 Natural lifetime
Very fast1-10 ps
Einstein A coefficient A21=1/τOscillator strength, f, and fluorescence lifetime τ
2122
3
21 8A
e
mcf
o
For band centered at 500 nm, Fully allowed transition has lifetime of 4 ns(for one electron)
Dyes has several valence electrons, larger fLifetimes between 1-4 ns
Fluorescent lifetime depends on environment:Fluorescent lifetime depends on environment:Used in microscopy as contrastUsed in microscopy as contrast
υυ=light frequency, m=mass of electron,=light frequency, m=mass of electron,c=speed of light, c=speed of light, ee= electron charge= electron charge
Unquenched emission:Normal QY, lifetime
Quenched emissionDecreased QY, lifetimee.g. metals, aggregation
Unquenched and Quenched Emission
iscf
f
kk
k
nonradiscf
f
kkk
k
Gold somewhat quenchesthe fluorescence
2 general approaches: time domain and frequency domain
Short pulse laser modulate CW laser
Frequency Domain Methods for Lifetime Measurements:Modulate laser and measure phase change of fluorescence
Use cw laser (e.g. argon ion)Modulate at rate nearInverse of emission lifetime10-100 MHz
Measure phase changewith Lockin amplifier
Modulation Methods in Frequency Domain
Modulate laser and ICCD(intensified CCD camera)Better S/N
Modulate laser
ICCD Detectors for Lifetime measurements:Frequency domain and some time-domain
Needs to be gated rapidlyWidefield imaging (no sectioning)High quantum yieldVery expensive $80KRegular CCDs:10-20K
Historically Most common
Microchannel platesAmplify signal ~10 fold
Time-domain Widefield Lifetime imaging with ICCD
Variable delayed gate or many gatesis scannedTo sample exponential decay:Many frames (for each delay)
ICCD has no time intrinsic response: slow readoutGated gain
Two-photon has short pulse laser for time-gating
French
Ti:sapphire
Higher viscosityShorter lifetimeBetter chance forNon-radiative decay
Time domain methods for lifetime measurementsWith gated electronics and fast detectors (not gain modulated)Best for point detection, PMT on laser scanning
Synchronized Gating done by pulsed laser (e.g. ti:sapphire laser)
Collect data from multiple gates (windows)At the same time, fit to exponential
PMT Detectors for Lifetime measurements
~300 picosecond resolutionBetter with deconvolutionCost ~$500
~30 picosecond resolutionNo dispersionCost ~$15000fragile
PMTS have low quantum yield(10-20%), MCP worse ~5%
Microchannel plate photomultiplier: full of holes, kick off electrons
Dispersion in time of flightacross 14 dynodesLimits time response
300-500 picosecond resolutionVery small area (200 sq microns)Not good for scanningHigh quantum yield (up to 70% at 700 nm)Low count rate (~10 MHz)$5KExtremely fragile!!
Avalanche Photodiode (APD)
Time gating measurements of fluorescence decayTemporal Resolution defined by IRF (laser, detector, electronics)
IRF=instrument response function,Must be (much) shorter than fluorescence lifetime (delta function)to avoid convolution
Measure IRF with reflectionor known short lifetimee.g. Rose Bengal (90 ps)
Ideal IRF Real IRF
Gate away from IRF (laser pulse, PMT response)Lose photons
Practical determinations of theInstrument Response Function
1) Laser modern lasers: ti:sapphires 100 femtosecondLifetimes: nanosecondsNot a factorWas 20-30 years ago before modelocked lasers
2) DetectorsAPD or PMT response ~200 picoseconds: can beMCP-PMT 30 ps: not typical limitation
3) TCSPC or gating Electronics20-50 ps (depending on sophistication) Can be convolved with MCP-PMT response
Time-correlated single photon counting:•most flexibility, most accurate,•samples whole decay•Best time response
Measures time of flight of photonsAfter excitation pulse
Bins data at each time intervalRather than gating
Collect enough photons to approximate exponential:
Slower than gating butBetter measurement, Can separate biexponentials: Multiple components
Principles of time-correlated single photon counting
TAC or TDC measures time of flight, bins photons
Been aroundFor decades
Time-Correlated Single Photon Counting electronicsOn laser scanning microscope (recent)
TCSPC electronics synchronized with laser scanning electronics:Pixel, line, frame synchHistorically very hard: mostly homebuilt (e.g. Gerritsen)
Becker &Hickel addon to Zeiss Laser scanning confocal
Electronics all in one PCI board, ~50K addon
Intensity vs fluorescence lifetime image
Same dye, different lifetime because of environment
Quenched close to Nucleus due toHigher concentrationLower lifetime
Intensity and lifetime measurements
CFP-YFP linked by short peptide chainEnergy is transferred from CFP to YFPLifetime reveals info intensity does not
Duncan, J. Microscopy 2004
TCSPC FLIM using ECFP
2 distinct lifetimes: meaning?
Performance of Frequency and time domain methods
TCSPC best for efficiency, S/NBut more expensive (ti:sapphire laser)But already have if have 2-photon microscope
Long Acquisition Times for TCSPC FLIM:Need enough data to approximate decay
Bright stains106/s
Dim stains104/s
May bleach before done imagingDetection with 2-4 gates may be better ifShort on photons
need100-100000Photons/pixel
GFP lifetime increasesWith increasing viscosity
Different lifetime for B cells at immuno Junction with natural killer (NK) cellEGFP::MHC
Autofluorescence of Rat Ear
Contains collagen, elastin :Single exponential not sufficient for multiple components
Fits to two discrete components noisy
Continuous lifetime distributionBetter for multiple components
Mean tauFor pixels
Width, h, of distributionFor pixel
Unless know componentsStretched exp is betterRepresentative of physiologyand provides more data
FLIM as Diagnostic of Joint Disorder
H&E staining
Widefieldfluorescence
WidefieldFLIM
Little info
Detail revealed by FLIM
Fixed, thin sections(few microns)
FLIM as Cancer Diagnostic
Benign
Carcinoma
H&E staining Widefield FLIM
FLIM shows morphology like H&E histologyCan optically section and no staining with FLIMWith 2-p can do thick tissues (few hundred microns)
More contrastThan H&E
ProbablyNADH, FAD
Widefieldfluorescence
WidefieldFLIM
FLIM Diagnostics of arterial plaque
Clear lifetimeDifference in Normal and plaque:Not visible by Fluorescence intensity
FLIM via endoscope as clinical toolWorks like through microscope
Lifetime of NADH, FAD changes from normal To cancer
White PNAS2007
DonorExcitation
Donor Emission
Donor Excitation
AcceptorEmission
Fluorescence Resonance Energy Transfer (FRET)
Donor emission overlaps with Acceptor Absorption:Highly distance dependent
FRET probes conformational changes
Different conformation givesDifferent FRET signature
FRET increasesIn both cases
Inter and IntramolecularForms of FRET withProteins
CFP-YFP good combo
Protein-Protein InteractionsIn cytoplasm andmembranes
No FRET forNo overlap of donor emission,acceptor absorption
When FRET Occurs
No FRET forOrthogonal dipoleorientation
No FRET for moleculesmore than 10 nm apartR0=distance where FRET=0.5
Typical Values of Ro
Donor Acceptor Ro (Å)
Fluorescein Tetramethylrhodamine 55
IAEDANS Fluorescein 46
EDANS DABCYL 33
Fluorescein Fluorescein 44
BODIPY FL BODIPY FL 57
Fluorescein QSY 7 dye 61
Cy3 Cy5 53
CFP YFP 50
green red
GFPs and other colored “FPs have transformed FRET microscopy
Before had to label proteins, then introduce
Number of FRET Publications since 1989
60
6
60
RR
RFRET
Fluorescence Resonance Energy Transfer -Detection of Probe Proximity
0
0
0
0
0
0
D
DD
AMax
A
AA
D
DD
FF
FF
F
FFFRET
R0 typically 40-50 Angstroms50% transfer
Practical Challenges to FRET Quantitation
• Emission from A contaminates D channel (filters)• Emission from D contaminates A channel• Unknown labeling levels for D and A• Signal variation due to bleaching
– Complicates kinetic studies– Bleaching rate of D can actually be slowed by FRET
Solutions:• Separately labeled D and A controls to define bleedthrough• Acceptor destruction by photobleaching to establish • Dual wavelength ratio imaging to normalize away variations in label levels and bleaching effects
0DF
Fluorescent Proteins as D-A PairsIssue of Spectral Overlap
Better overlap,FRETBut more bleedthrough
Poor Spectral overlap,But less bleedthrough
Survey of FRET-Based Assays
• Protease activity
• Calcium Ion measurements
• cAMP
• Protein tyrosine kinase activity
• Phospholipase C activity
• Protein kinase C activity
• Membrane potential
Principle of Operation of Chameleon Calcium Indicators
FRET Increases when CaM binds Calcium ionsConformation changes, CFP-YFP closer together
Potential Sensor Based on FRET
Mechanism and Single CellsGonzalez and Tsien, Biophys J., 1995
Demonstration on Leech GanglionKleinfeld, et al., Neuron, 1999
Improved indicatorsGonzalez JE, Tsien RY. 1997.
Chemistry and Biology 4:269-277.
Donor= Di4-ANEPPSFast voltage sensor
Acceptor=OxonolSlow voltage sensorFRET pair more sensitive
Lifetime and FRET
Large change in lifetime for quenched donor upon FRET
0
0
0
0
0
0
D
DD
AMax
A
AA
D
DD
FF
FF
F
FFFRET
FRET should have bi-exponential decay, quenched and unquenched:Long and short lifetime components
CFP and YFP FRET by Lifetime Imaging
Channel changes conformation, distance changes, Donor quenching occurs due to FRET
Short lifetime is FRET from DonorFor given pixel Ratio of fast to slow decay coefficientsis estimate of FRET efficiency
Duncan, J. Microscopy 2004
CFP and YFP tethers FRET by Lifetime Imaging
Donor Lifetime goes up post acceptor bleaching
FRET Outcomes
Donor decreases
Acceptorincreases
Donor lifetimedecreases
Donor fluorescenceAnisotropy increasesAcceptor decreases
FRET pair anisotropy
Donor Anisotropy Increases: shorterLifetime, less likely to Rotate before emission
Extent of depolContains relativeorientation
Emission dipole usuallyParallel to excitation dipole:FRET to other orientationDepolarizes acceptor emissionNot constrained by laser
Much better dynamic rangeThan lifetime based changes ~10x
Anisotropy measurement more accurate
Piston, BJ2004
Fluctuation (fluorescence) Correlation Spectroscopy (FCS)
Fluctuations in excitation volumedue to Diffusion, reactions
Diffusion times for globular proteins
0
100
200
300
400
500
0 200000 400000 600000 800000 1000000
molecular weight (kD)diff
usi
on t
ime (
mic
rose
cond
s)
Spherical molecule
Diffusion coefficient
Diffusion Demonstration
http://physioweb.med.uvm.edu/diffusion/FrapPages1.htm
Normalized Autocorrelation Function
Compares probability of detecting photon at time t with some latter time t + τ
Autocorrelation the expected value of the product of a random variable or signal realization with a time-shifted version of itself
FCS: autocorrelation of the fluorescence photons of one and the same molecule.
FCS decay: diffusion, bindingMaximum at tau=0Small tau: large correlationLarge tau: tend to 0
Form for translational diffusion
N=concentration of molecules in focal volume
τD =diffusion time, R=ωz/ωxy of observation volume
Need to measure Point Spread FunctionTo determine observation volume
Determination of Point Spread Function of Microscope
NAd
2
22.1min
Abbe` Limit
175 nm BeadSub-resolution
Volume is EllipsoidAxial ~NA2
PSF and Beam Waist
Imaging sub-resolution 100 nm fluorescent beads
Use 1/e2 points to get ω beam waist (87%)
D =2/4D
8D for two-photon
webb
FCS of Rhodamine in Sucrose Solution
Higher concentrationsShorter correlation times
1/y=number of moleculesIn focal volume
webb
Molecular interactions
FCS is a method to study molecular interactions between – fluorescence-tagged ligands and target molecules such as receptors, – unidentified, untagged compounds and tagged ligands
in competitive binding – protein-protein interactions – individual events of signal transduction cascades within cells.
Concentration range
FCS allows molecular interactions to be characterized over a wide dynamic range-concentrations of labeled particles between 200 nM to 200 pM -can be used. Because of the ultra-lowmeasuring volume of 10-15 l required for FCS measurement, nanoliter to microliter sample volumes are sufficient.
Physical state
Differences in the physical state of the molecule of interest, such as bound vs. free, cleaved vs. intact,can be discerned by FCS provided that the variant forms differ sufficiently in their size-related diffusion – peptides bound to soluble receptors, – ligands bound to membrane-anchored receptors, – viruses bound to cells, – antibodies bound to cells, – primers bound to target nucleic acids, – regulatory proteins /protein-complexes in interaction
with target DNA or RNA – enzymatic products.
If the diffusion properties of the reactants are too similar, both reactants have to be labeled withfluorescent dyes with different excitation and emission spectra.
Mathematical model for autocorrelation
Two component autocorrelation curve
Diffusion of structures > 50 nm may be hindered in cytoplasmtau values > 1 ms may not represent free diffusion
Binding to immobile species,e.g. microtubules
Binding to mobile receptor Binding to immobile receptor Motility along microtubule
ConcentrationDiffusion of receptor
ConcentrationKd
On rate (M-1sec-1)Off rate (sec-1)
Mobile/immobileMean squared displacement
The slow component in living cells
The slow component for hnRNP A2 (tau = 21 msec) may represent granule movement on microtubules
Measuring dissociation constant by FCS
Model:
ACCA
ABBAKd
Kd
Where A is a protein with low mobility, B is a mobile fluorescently labeled protein, and Cis endogenous, unlabeled version of B. Both B and C bind to A with the same affinity.
When B binds to A, its mobility changes. This can be observed by FCS as twocomponents with diffusion times 1 and 2 and amplitudes y1 and y2.
Equations
21
2
*
*
yy
yR
AC
CAKd
AB
BAKd
ACCCt
ABBBt
ACABAAt
Where At, Bt, and Ct are total concentrations of proteins A, B, and C correspondingly.
The dissociation constant can be expressed from the equations as
RCtBtR
AtKd
1
tau2= off rate
Carson, JMB
Need to measureEndogenous, addedProtein concentrations
Determine taus,Mobile fractionsBy bleaching
fluorescentmolecules
Cross-correlation spectroscopy
3D Gaussianconfocal detection volume
~1 femtoliter
diffusiontrajectories
Individual fluorescent molecules are detected as single channel photon count fluctuations. Bound
molecules are detected as coincident dual channel fluctuations.
Cross-correlation analysis provides a measure of the
number and rate of diffusion of bound molecules.
Cross-correlation function Grg(t) = < Ig(t).Ir(t+t) >
1
1.02
1.04
1.06
1.08
1.1
10 100 1000 10000
microseconds
Alexa488 RNASyto61cross-correlation
Dual channel fluctuation
10000
15000
20000
25000
30000
35000
40000
45000
50000
0 1 2 3 4 5 6 7 8 9 10
seconds
Alexa488 RNASyto61
Count rate
Photon Counting Histogram
Same raw data as autocorrelation
Measure of probability of brightness at given time
Allows determination of dimerization or oligerimization
By diffusion, assuming hardspheresDimer would increase τD
By only 26%. Cannot measure by FCS
However brightness would increase by 2x
PCH can determine concentrations
Gratton, BJ
PCH can differentiate fluorophores based on brightness