1 ken hanson mwf 9:00 – 9:50 am office hours mwf 10:00-11:00 chm 5175: part 2.6 time-resolved...
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1
Ken HansonMWF 9:00 – 9:50 am
Office Hours MWF 10:00-11:00
CHM 5175: Part 2.6Time-resolved emission
Source
hn
Sample Detector
Clock
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Steady-state Emission
550 600 650 700 750 800 8500
50000
100000
150000
200000
250000
Inte
nsi
ty
Wavelength (nm)
Intensity vs. WavelengthSource Samplehn
Information about emission intensity (yield) and wavelength.
S0
S1
EnergyConstant Excitation
Constant Emission
Equilibrium between absorption, non-emissive decay and emission.
Non-emissive
decay
hn
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Time-resolved Emission
Intensity vs. Time
Information about emission lifetimes.
S0
S1
EnergyPulsed
Excitationkrknr
0 200 400 600 800 10000
1000
2000
3000
4000
5000
Inte
ns
ity
Time (ns)
Short Burst of Light
Competition between non-emissive decay and emissive rates.
Source Samplehn
hn
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Single Molecule Emission
Excited State Lifetime of an individual molecule: 0 – infinity
Anthracene Excited state Lifetime:Time spent in the excited state (S1) prior to radiative (kr) or non-radiative decay. (kr)
Ex Em
S0
S1
Energy
Time
Ex Em Ex
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Ensemble Emission
Time-resolved EmissionIntensity vs. Time
0 200 400 600 800 10000
1000
2000
3000
4000
5000
Inte
ns
ity
Time (ns)
Single Molecule Emission
Excited State Lifetime of an individual molecule: 0 – infinity
Observe many single molecule emission events!
Ex Em
S0
S1
Energy
Time
Ex Em Ex
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Ensemble Emission
hn Time 1
64 excited states32 excited states
+ 32 photons
Time 2
Time 3
16 excited states + 16 photons
Time 4
8 excited states + 8 photons
4 excited states + 4 photons
Time 5
etc.
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Ensemble Emission
hn Time 1
64 excited states32 excited states
+ 32 photons
Time 2
Time 3
16 excited states + 16 photons
Time 4
8 excited states + 8 photons
4 excited states + 4 photons
Time 5
etc.
0 2 4 6 8 100
10
20
30
40
50
60
70
# E
xcit
ed S
tate
s
Time
0 1 2 3 4 5 6 7 8 9 100
10
20
30
40
Em
issi
on
Inte
nsi
ty
Time
32 photons
16 photons
8 photons
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kr + knr
Excited State Decay Curve
/t*
*
e)0(n
)t(n
0 2 4 6 8 100
10
20
30
40
50
60
70
# E
xcit
ed S
tate
s
Time
n*(0) is the # of the excited state at time 0
n*(t) is the # of the excited state at time t
t is the lifetime of the excited state
S0
S1
EnergyPulsed
Excitationkrknr
t =1
We don’t get to count the number of excited state molecules!
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Intensity Decay Curve
I(0) is the initial intensity at time zero
I(t) is the intensity at time t
t is the lifetime of the excited state0 1 2 3 4 5 6 7 8 9 10
0
10
20
30
40
Em
issi
on
Inte
nsi
ty
Time
= e-t/t
kr + knr
t =1
t = time it takes for 63.2 % of excited states to decay
t should always be the same for a given molecule under the same conditions
0 200 400 600 800 10000
1000
2000
3000
4000
5000
Inte
nsi
ty
Time (ns)
I(t)
I(0)
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time
intensity
1.00 --
1/e
Exciting pulse
Emission
time
Log intensity
Exciting pulse
Emission
Intensity Decay Curve
Linear Scale Log Scale
= e-t/tI(t)
I(0)
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Spectra Decay
= e-t/tI(t)
I(0)
inte
nsity
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Why do we care about lifetimes?• Electron transfer rates
• Energy transfer rates
• Distance dependence
• Distinguish static and dynamic quenching
• Fluorescence resonance energy transfer (FRET)
• Track solvation dynamics
• Rotational dynamics
• Measure local friction (microviscosity)
• Track chemical reactions
• kr and knr (if you know F)
• GFP- Nobel prize, expression studies
• Sensing
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Lifetime Measurements
Inte
nsity
time
Light source
Time Domain
Pulsed MethodHarmonic or phase-modulation method
Frequency Domain
time
Inte
nsity
Light source
Source Samplehn
hn
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Samplehn
hn
hn h
n
Low I0 Excitation
High I0 Excitation
hnhn
Low I0 Excitation
Time
Frequency-domain Method
I0
Measure Events with Respect to Frequency
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Frequency-domain Method
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Frequency-domain Method
Excitation Modulation = ab
a = average intensity
b = average-to-peak intensity
Emission Modulation = AB
A = average intensity
B = average-to-peak intensity
Modulation (m) =
(B/A)(b/a)
Phase Shift (f)
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Frequency-domain Method
Modulation (m)
Phase Shift (f)
Ex Frequency ()
2/121 ]1)/1[( mm
tan1
Changing , measuring m and to calculate lifetime.
Phase (τφ) and modulation (τm) lifetimes
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tan12/121 ]1)/1[( mm
Frequency-domain Method
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Frequency-domain Method
• Lifetimes as short as 10 picoseconds • Can be measured with a continuous source• Tunable from the UV to the near-IR• Frequency domain is usually faster than time domain (same source)
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Frequency-domain Method
2/121 ]1)/1[( mm
tan1
f
m
Modulation (m)
Phase Shift (f)
Ex Frequency ()
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Frequency-domain Instrument
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Frequency-domain Method
List of Commercially Available Frequency-domain Instruments
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Lifetime Measurements
Inte
nsity
time
Light source
Time Domain
Pulsed MethodHarmonic or phase-modulation method
Frequency Domain
time
Inte
nsity
Light source
Source Samplehn
hn
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Inte
nsity
time
Light source
Time-Domain Method
• Pulsed method• Lifetimes as short as 50 fs• Multiple measurement techniques• Sources typically not as tunable as frequency domain
Emission
Emission intensity is measured following a short excitation pulse
Measure Events with Respect to Time
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Time-domain Techniques
1 s1 ms1 ms
1 ns1 ps1 fs
secondsmillimicronanopicofemto0.001 s0.000001 s0.000 000 001 s
0.000 000 000 001 s0.000 000 000 000 001 s
1 s
Excitation
PhosphorescenceFluorescence
Internal Conversion
Intersystem Crossing
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TCSPC
Time-domain Techniques
1 s1 ms1 ms
1 ns1 ps1 fs
Streak Camera MCSStrobeUp-conversion
Real-time Measurement
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Time-domain Techniques
1. Real-Time lifetime measurement (t > 200 ps)
2. Multi-channel scaler/photon counter (t > 1 ns)
3. Strobe –Technique (t > 250 ps)
4. Time-correlated single-photon counting ( t > 20 ps)
5. Streak-camera measurements (t > 2 ps)
6. Fluorescence up-conversion (t > 150 fs)
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Real-Time Lifetime
hn
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Real-Time Lifetime
Source
hn
Sample Monochromator
Detector
Clock
(1) (2)
(3)
(4)
1) Pulsed excitation
2) Sample excitation/emission
3) Monochromator
4) Detector signal
5) Plot Signal vs. Time
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Real-Time Lifetime
Light source D
etec
tor C
urre
nt
time
Emission
SourcesFlashlampLaserPulsed LED
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Real-Time Lifetime
• Make excitation pulse width as short as possible• Time resolution is usually detector dependent• Excited-state lifetime > IRF• Lifetimes > 200 ps
Instrument Response Function (IRF)
Det
ecto
r Cur
rent
time
Emission
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Real-Time Lifetime
0 200 400 600 800 10000
1000
2000
3000
4000
5000
Inte
nsi
ty
Time (ns)
100 averages
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Strobe-Technique
25 images per second
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Strobe-Technique
Photon Technology International (PTI)
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Strobe-Technique
time
Light PulseMeasurement Window
time
Light PulseMeasurement Window
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Strobe-Technique
time
Light Pulse
Measurement Window
time
time
Det
ecto
r Si
gnal
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Strobe-TechniqueStrobe-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
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(1) (2)
(3) (4)
(5)
Strobe-Technique
1) Trigger Signal
2) Excitation Flash
3) Detector Signal Delay
4) Detect
5) Output t > 250 ps
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Time-Correlated Single-Photon Counting (TCSPC)
Excited State Lifetime of an individual molecule: 0 – infinity
The sum an individual molecule lifetimes = t
Ex Em
S0
S1
Energy
Time
Ex Em Ex
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Low excitation intensity:
- Low number of excited state
- 20-100 pulses before emission is detected
- Only one or 0 photons detected per pulse
- Simulated single molecule imaging
Time-Correlated Single-Photon Counting (TCSPC)
Time
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1) Pulsed source “starts” the timing electronics
2) Timer “stopped” by a signal from the detector
3) The difference between start and stop is sorted into “bins.” -Bins are defined by a Dt after pulse at t = 0
Time
Detector Bins
Time-Correlated Single-Photon Counting (TCSPC)
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Time-Correlated Single-Photon Counting (TCSPC)
Time
Detector Bins
Sum the Photons per Bin
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Time-Correlated Single-Photon Counting (TCSPC)
Probability Distribution
Repeat
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Excitation Pulse
Time-Correlated Single-Photon Counting (TCSPC)
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Repeat: 10,000 counts in the peak channel
Time-Correlated Single-Photon Counting (TCSPC)
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Time-Correlated Single-Photon CountingSource:
Flash lampsolid state LED laser
Start PMT
Stop PMTsample
exc. monochromator
emis
sio
n
mo
no
chro
mat
or
pulsed source
t
1) Pulsed excitation (10kHz)
2) Monochromator
3) Beam Splitter
1) to trigger PMT
2) to sample
4) Excite Sample
5) Sample emits into monochromator
6) Emission hits PMT and timer stops
7) Repeat a million times
(1)
(3)
(2)
(4)(5)
(6)
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constant function discriminator (CFD)time-to-amplitude converter (TAC)programmable gain amplifier (PGA)analog-to-digital converter (ADC)
TCSPC
1) Pulsed excitation2) Ex CFD triggers TAC3) TAC voltage rises4) Em CFD stops TAC5) TAC discharges to PGA6) PGA siganl to ADC for a single data point
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48
TCSPC
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Advantages:– High sensitivity
– Large dynamic range (3-5 decades)
– Well defined statistics
– Temporal resolution down to 20 ps
– Very sensitive (low emission materials)
– Time resolution limited by detector
– Price as low as $15 K
Disadvantages:– “Long” time to acquire data
– Complicated electronics
– Stray light
– Lifetimes < 10 ms
– Resolution vs. acquisition time
TCSPC
Molecule with a 10 ms lifetime• 10,000 peak counts• 1024 bins for a 20 ms window• Total counts = 4,422,800• 20 ms rep rate• 1 count per 20 reps= 20.5 day measurement
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Acquisition Time
Time
Detector Bins
Resolution vs. Acquisition Time
5 ns wide bin = 5 ns resolution10 minutes to acquire 10,000 counts
Time
Detector Bins
1 ns wide bin = 1 ns resolution50 minutes to acquire 10,000 counts
Resolution
Acquisition TimeResolution
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Time
Repetition Rate to High
hn
hn
Real start-stop-time
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Sign
al
time
Repetition Rate to High
If the rep rate is too high the histogram is biased to shorter times!
Measured t < Real t
Keep rep rate at least 10 times slower than your t
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Stop count rate < 2% of the excitation rate.
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.
Intensity to High
Single Photon Counting only counts the first photon!
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Photoelectric Effect
Photon Energy - binding energy = electron kinetic energy
Side Note: PMT Lifetime
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Side Note: PMT LifetimePhotoelectric Effect
Photon Energy - binding energy = electron kinetic energy
Higher Energy Photons = Faster Signal
Measured Lifetime < Real Lifetime
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Streak-CameraTemporal profile from Spatial profile
Laser Pointer Duty Cycle Calculating Duty Cycle
Pointer Motionm/s
Dist
ance Length
(spatial)
Use length to calculate time
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Streak-CameraCathode Ray Tube
e-
+
-
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Streak-Camera
(2)(1)
1) Light hits cathode (ejects e-)
2) Voltage sweep from low to high
3) e- hits MCP-Phosphor Screen
4) Emitted photos hit CCD detector
Source
hn
Sample Monochromator
(3)
(4)
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Calculating Duty Cycle
Pointer Motionm/s
Dist
ance Length
(spatial)
Use length to calculate time
Streak-Camera
Sweep Ratem/s
Length
Use length and intensity to calculate lifetimee-
time(0) time(t)
0 200 400 600 800 10000
1000
2000
3000
4000
5000
Inte
ns
ity
Time (ns)
+-
Intensity
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Streak-Camera
(2)(1)
1) Light hits cathode (ejects e-)
2) Voltage sweep from low to high
3) e- hits MCP-Phosphor Screen
4) Emitted photos hit CCD detector
Source
hn
Sample Monochromator
(3)
(4)
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Electrons that arrive first hit the detector at a different position compared to electrons that arrive later.
Streak-Camera
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Streak-Camera
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• Advantages:– Direct two-dimensional resolution– Sensitivity down to single photon– Very productive– Not detector limited (like TCSPC)
• Disadvantage: – Depends on high stability of laser– Limited time resolution: 2-10 ps– Needs careful and frequent calibration– Expensive
Streak-Camera
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Streak-Camera
Time resolution down to 2ps or even 100s of femtoseconds.
TCSPC
Instrument Response Functions
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Fluorescence up-conversion
Sum Frequency Method
ωsum = ω1 + ω2
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Fluorescence up-conversion
1) Excitation pulse/gate pulse
2) Sample is excited
3) Sample Emission
4) Emission and Gate are collinear
5) NLO crystal sums Emission and Gate
6) Only Summed Light is measured
(1)
(4)
(2)
excitation beamgate beam
(1)
(3)
(5)(6)
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Fluorescence up-conversionExcitation pulse
Emission
Inte
nsity
Excitation pulse
Inte
nsity
Gatepulse
td1
Inte
nsity
Summed Light at time 1
time
time timeExcitation
pulse
Inte
nsity
Gatepulse
td2
Inte
nsity Summed Light
at time 2
time time
Inte
nsity
time
Control td and measure only summed light
Graph of td vs intensity
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Fluorescence up-conversion
1) Excitation pulse/gate pulse
2) Sample is excited
3) Sample Emission
4) Emission and Gate are collinear
5) NLO crystal sums Emission and Gate
6) Only Summed Light is measured
(1)
(4)
(2)
excitation beamgate beam
(1)
(3)
(5)(6)
Signal is only measured when gate is pulsed
td is controlled by the delay track
Light Travels 0.9 m in 1 ns
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ComparisonIn
tens
ity
time
Control td and measure only summed light
Detector is not time resolved (left open).Not limited by detector speed.Data point limited by pulse width (fs)
Sum Frequency Generation TCSPC
Detector Bins
Inte
nsity
time
Limited by detector response.Data point limited by PMT (10 ps)
Control excitation measure td
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Fluorescence up-conversion
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Phys . Chem. Chem. Phys. 2005, 7, 1716 – 1725.
Fluorescence up-conversion
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Fluorescence up-conversion
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74
• Advantage: – (very) high time resolution, limited mainly by laser pulse duration
• Disadvantages:– Demanding in alignment– Limited sensitivity, decreasing with increasing time resolution
(crystal thickness)– Required signal calibration
Fluorescence up-conversion
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Decay Fitting
Exponential decay
= e-t/tI(t)
I(0)
Non-exponential decay
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Non-exponential Decay (Log)
Exponential decay Non-exponential decay
Time Time
Inte
nsity
= e-t/tI(t)
I(0)
Inte
nsity
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Non-exponential
Possible explanations:
- Two or more emitters
- In homogeneous samples (QDs)
- Dual Emission
- Multiple emissive sites
On surfaces
Polymer Films
Peptides
Dual Emission
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Intensity
t / ns
Log
I
t / ns
5 ns
50 ns
Non-exponential Decay
= A1e-t/ 1t + A2e-t/ 2tI(t)
I(0)
A1 = amplitude of component 1t1 = lifetime of component 1A2 = amplitude of component 2t2 = lifetime of component 2
Linear Scale
Log Scale
Biexponential Fit
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Non-exponential Decay
= A1e-t/ 1t + A2e-t/ 2tI(t)
I(0)
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Limitations of Multi-exponential Fits
Linear Scale: No differenceLog Scale: minor differences at 30–50 ns
At 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.
Biexponential Fits
t1 = 5.5 ns and t2 = 8.0 nsor
t1 = 4.5 ns and t2 = 6.7 ns
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Fitting Data
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y = A1e-k1t + A e-k2t + A3e-k3t
c2 = 26.466
y = A1e-k1t + A e-k2t
c2 = 2.133
The Data Exponential
Bi-exponential Tri-exponential
c2 = 1.194
Multi-exponential Fits
y = A1e-k1t
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J. of Political Economy 2005, 113, 949
It could be worse!
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Time-resolved Emission End
Any Questions?