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

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

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nonradiscf

f

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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 10

Natural lifetime

Very fast1-10 ps

Einstein A coefficient A21=1/τOscillator strength, f, and fluorescence lifetime τ

2122

3

21 8A

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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

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f

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nonradiscf

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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 (100 to 10 ns)

Measure phase changewith Lockin amplifier

Modulation Methods in Frequency Domain

Modulate laser and ICCD(intensified CCD camera)Better S/N for imaging

Modulate laserOK for spectroscopy

ICCD Detectors for Lifetime measurements:Frequency domain and some time-domain

Needs to be gated rapidlyWidefield imaging (no sectioning)High quantum yieldVery expensive $20-80KRegular CCDs:10-20K

Historically Most common

Microchannel platesAmplify signal ~10 fold

Time-domain Widefield Lifetime imaging with ICCDVariable delayed gate or many gatesis scannedTo sample exponential decay:Many frames (for each delay)

ICCD has no time intrinsic response: slow readout gated gain.Use laser pulse width much less than fluorescence lifetime

Two-photon scope has short pulse laser for time-gating

FrenchOptics express

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 limiting governing 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

Mark Terasaki Flash animation

http://www.terasaki.us/flash/lab/flim01.swf  

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?

GFP lifetime increasesWith increasing viscosityLimits motion, nonrad

Different lifetime for B cells at immuno Junction with natural killer (NK) cellEGFP::MHC

Performance of Frequency and time domain methods

TCSPC best for efficiency, S/N, information contentBut 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

Autofluorescence of Rat Ear

Contains collagen, elastin :Single exponential not sufficient for multiple components

Fits to two discrete components noisy (large residuals) French, 2001, Biophys J.

Time domainWith gated ICCD

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

Widefieldautofluorescence

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 and high to low-grade

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

RRRFRET

Fluorescence Resonance Energy Transfer -Detection of Probe Proximity

0

0

0

0

0

0

D

DD

AMax

A

AA

D

DD

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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

Want sharp filters, But throw away photons

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

FFFF

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FRET should have bi-exponential decay, quenched and unquenched:Short and long 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

With FRET: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