2Steady-state Emission SampleSourceIntensity vs. WavelengthhnhnS0S1EnergyNon-emissive decayConstant ExcitationConstant EmissionEquilibrium between absorption, non-emissive decay and emission.Information about emission intensity (yield) and wavelength.
3Time-resolved Emission Information about emission lifetimes. SampleSourcehnIntensity vs. TimehnShort Burst of LightS0S1EnergyPulsed ExcitationkrknrCompetition between non-emissive decay and emissive rates.Information about emission lifetimes.
4Single Molecule Emission Excited state Lifetime:Time spent in the excited state (S1) prior to radiative (kr) or non-radiative decay. (kr)AnthraceneS1EnergyExEmExEmExS0TimeExcited State Lifetime of an individual molecule: 0 – infinity
5Ensemble Emission Time-resolved Emission Intensity vs. Time Single Molecule EmissionExcited State Lifetime of an individual molecule: 0 – infinityExEmS0S1EnergyTimeObserve many single molecule emission events!
6Ensemble Emission 64 excited states hn Time 1 Time 2 Time 4 Time 3 + 32 photons64 excited stateshnTime 1Time 2Time 4Time 34 excited states+ 4 photons8 excited states+ 8 photons16 excited states+ 16 photonsTime 5etc.
8Excited State Decay Curve EnergyPulsed Excitationkrknrn*(0) is the # of the excited state at time 0n*(t) is the # of the excited state at time tt is the lifetime of the excited state1t=kr + knrWe don’t get to count the number of excited state molecules!
9I(t) = e-t/t I(0) Intensity Decay Curve t 1 = kr + knr I(0) is the initial intensity at time zeroI(t) is the intensity at time tt is the lifetime of the excited statekr + knrt=1t = time it takes for 63.2 % of excited states to decayt should always be the same for a given molecule under the same conditions
12Why do we care about lifetimes? Electron transfer ratesEnergy transfer ratesDistance dependenceDistinguish static and dynamic quenchingFluorescence resonance energy transfer (FRET)Track solvation dynamicsRotational dynamicsMeasure local friction (microviscosity)Track chemical reactionskr and knr (if you know F)GFP- Nobel prize, expression studiesSensing
19Frequency-domain Method Lifetimes as short as 10 picosecondsCan be measured with a continuous sourceTunable from the UV to the near-IRFrequency domain is usually faster than time domain (same source)
20Frequency-domain Method Ex Frequency ()Modulation (m)Phase Shift (f)fm
24Time-Domain Method Intensity time Measure Events with Respect to Time Light sourceIntensitytimeEmission intensity is measured following a short excitation pulseEmissionPulsed methodLifetimes as short as 50 fsMultiple measurement techniquesSources typically not as tunable as frequency domain
29Real-Time Lifetime (4) (3) 1) Pulsed excitation SourceClock(1)(2)(3)(4)hnDetectorSampleMonochromator1) Pulsed excitation2) Sample excitation/emission3) Monochromator4) Detector signal5) Plot Signal vs. Time
30Detector Current time Real-Time Lifetime Emission Light source Sources FlashlampLaserPulsed LED
31Instrument Response Function (IRF) Real-Time LifetimeDetector CurrenttimeEmissionInstrument Response Function (IRF)Make excitation pulse width as short as possibleTime resolution is usually detector dependentExcited-state lifetime > IRFLifetimes > 200 ps
34Strobe-TechniquePhoton Technology International (PTI)
35Strobe-Technique Light Pulse time Light Pulse time Measurement Window
36Strobe-Technique Light Pulse time time Detector Signal time Measurement WindowtimeDetector Signal
37Strobe-Technique Strobe-Technique TCSPC “Full decay curve is attainable after just one sweep (100 pulses)”“TCSPC: for every 100 pulses, you get only up to three useful points”“The Strobe technique is much faster than the TCSPC technique for generating the decay curve. This is particularly important in the life science area. Whereas the chemist can take hours or days to measure an inert chemical very accurately, the life scientists’ cell samples are long dead. “Lower Time Resolution
38Strobe-Technique (2) (1) (4) (3) 1) Trigger Signal (5) 2) Excitation Flash3) Detector Signal Delay4) Detect5) Outputt > 250 ps
39Time-Correlated Single-Photon Counting (TCSPC) EmExEnergyExEmExS0TimeExcited State Lifetime of an individual molecule: 0 – infinityThe sum an individual molecule lifetimes = t
40Time-Correlated Single-Photon Counting (TCSPC) Low excitation intensity:- Low number of excited statepulses before emission is detected- Only one or 0 photons detected per pulse- Simulated single molecule imagingTime
41Time-Correlated Single-Photon Counting (TCSPC) 1) Pulsed source “starts” the timing electronics2) Timer “stopped” by a signal from the detector3) The difference between start and stop is sorted into “bins.”-Bins are defined by a Dt after pulse at t = 0Detector BinsTime
42Time-Correlated Single-Photon Counting (TCSPC) Sum the Photons per BinDetector BinsTime
43Time-Correlated Single-Photon Counting (TCSPC) RepeatProbability Distribution
45Time-Correlated Single-Photon Counting (TCSPC) Repeat: 10,000 counts in the peak channel
46Time-Correlated Single-Photon Counting emission monochromator Source:Flash lampsolid state LEDlaser1) Pulsed excitation (10kHz)2) Monochromator3) Beam Splitter1) to trigger PMT2) to sample4) Excite Sample5) Sample emits into monochromator6) Emission hits PMT and timer stops7) Repeat a million timespulsed source(1)(2)exc. monochromatorStart PMTt(3)(3)Stop PMTemission monochromatorsample(4)(6)(5)
47TCSPC 1) Pulsed excitation 2) Ex CFD triggers TAC 3) TAC voltage rises 4) Em CFD stops TAC5) TAC discharges to PGA6) PGA siganl to ADC for a single data pointconstant function discriminator (CFD)time-to-amplitude converter (TAC)programmable gain amplifier (PGA)analog-to-digital converter (ADC)
49TCSPC Advantages: Disadvantages: High sensitivity Large dynamic range (3-5 decades)Well defined statisticsTemporal resolution down to 20 psVery sensitive (low emission materials)Time resolution limited by detectorPrice as low as $15 KDisadvantages:“Long” time to acquire dataComplicated electronicsStray lightLifetimes < 10 msResolution vs. acquisition timeMolecule with a 10 ms lifetime10,000 peak counts1024 bins for a 20 ms windowTotal counts = 4,422,80020 ms rep rate1 count per 20 reps= 20.5 day measurement
50Resolution vs. Acquisition Time Detector BinsDetector BinsTimeTime5 ns wide bin = 5 ns resolution10 minutes to acquire 10,000 counts1 ns wide bin = 1 ns resolution50 minutes to acquire 10,000 countsResolutionAcquisition TimeResolutionAcquisition Time
51Repetition Rate to High hnhnRealstart-stop-timeTime
52Repetition Rate to High SignaltimeIf the rep rate is too high the histogram is biased to shorter times!Measured t < Real tKeep rep rate at least 10 times slower than your t
53Stop count rate < 2% of the excitation rate. Intensity to HighSingle Photon Counting only counts the first photon!Limited number of emitted photons. Failure to do so can lead to a biasing towards detection of photons arriving at shorter times, a phenomenon known as pulse pile up.Stop count rate < 2% of the excitation rate.
54Side Note: PMT Lifetime Photoelectric EffectPhoton Energy - binding energy = electron kinetic energy
55Side Note: PMT Lifetime Photoelectric EffectPhoton Energy - binding energy = electron kinetic energyHigher Energy Photons = Faster SignalMeasured Lifetime < Real Lifetime
56Temporal profile from Spatial profile Streak-CameraTemporal profile from Spatial profileLaser Pointer Duty CycleCalculating Duty CycleLength (spatial)DistancePointer Motionm/sUse length to calculate time
64Streak-Camera Advantages: Disadvantage: Direct two-dimensional resolutionSensitivity down to single photonVery productiveNot detector limited (like TCSPC)Disadvantage:Depends on high stability of laserLimited time resolution: 2-10 psNeeds careful and frequent calibrationExpensive
65Streak-Camera Instrument Response Functions TCSPC Time resolution down to 2ps or even 100s of femtoseconds.
66Fluorescence up-conversion Sum Frequency Methodωsum = ω1 + ω2
67Fluorescence up-conversion (1)(4)(2)excitation beamgate beam(3)(5)(6)1) Excitation pulse/gate pulse2) Sample is excited3) Sample Emission4) Emission and Gate are collinear5) NLO crystal sums Emission and Gate6) Only Summed Light is measured
68Fluorescence up-conversion Excitation pulseGraph of td vs intensityIntensityEmissionIntensitytimetimeExcitation pulseGatepulseSummed Light at time 1td1IntensityIntensitytimetimeExcitation pulseGatepulseSummed Light at time 2td2IntensityIntensitytimetimeControl td and measure only summed light
69Fluorescence up-conversion (1)(4)(2)excitation beamgate beam(3)(5)(6)1) Excitation pulse/gate pulse2) Sample is excited3) Sample Emission4) Emission and Gate are collinear5) NLO crystal sums Emission and Gate6) Only Summed Light is measuredSignal is only measured when gate is pulsedtd is controlled by the delay trackLight Travels 0.9 m in 1 ns
70Control excitation measure td ComparisonSum Frequency GenerationTCSPCDetector BinsIntensityIntensitytimetimeControl td and measure only summed lightControl excitation measure tdDetector is not time resolved (left open).Not limited by detector speed.Data point limited by pulse width (fs)Limited by detector response.Data point limited by PMT (10 ps)
74Fluorescence up-conversion Advantage:(very) high time resolution, limited mainly by laser pulse durationDisadvantages:Demanding in alignmentLimited sensitivity, decreasing with increasing time resolution (crystal thickness)Required signal calibration
80Limitations of Multi-exponential Fits Biexponential FitsLinear Scale: No differenceLog Scale: minor differences at 30–50 nst1 = 5.5 ns and t2 = 8.0 nsort1 = 4.5 ns and t2 = 6.7 nsAt 50 ns there are only about 3 photons per channel with a 1-ns width. The difference between the two decays at long times is just 1–2 photons.