Time-resolved Emission

Report
CHM 5175: Part 2.6
Time-resolved emission
Source
Clock
hn
Sample
Detector
Ken Hanson
MWF 9:00 – 9:50 am
Office Hours MWF 10:00-11:00
1
Steady-state Emission
Sample
Source
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Intensity vs. Wavelength
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250000
Intensity
200000
S1
Energy
Constant
Excitation
Nonemissive
decay
Constant
Emission
150000
100000
50000
0
550
600
650
700
750
800
850
Wavelength (nm)
S0
Equilibrium between absorption, non-emissive decay and emission.
Information about emission intensity (yield) and wavelength.
Time-resolved Emission
Sample
Source
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Intensity vs. Time
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5000
Short Burst
of Light
Intensity
4000
S1
3000
2000
1000
Energy
Pulsed
Excitation
knr
kr
0
0
200
400
600
Time (ns)
S0
Competition between non-emissive decay and emissive rates.
Information about emission lifetimes.
800
1000
Single Molecule Emission
Excited state Lifetime:
Time spent in the excited state
(S1) prior to radiative (kr) or nonradiative decay. (kr)
Anthracene
S1
Ex
Energy
Em
Em
Ex
Ex
S0
Time
Excited State Lifetime of an individual molecule: 0 – infinity
Ensemble Emission
Time-resolved Emission
Intensity vs. Time
Single Molecule Emission
5000
4000
Intensity
Excited State Lifetime of an
individual molecule: 0 – infinity
3000
2000
1000
S1
0
Ex
Energy
Em
Em
Ex
Ex
0
200
400
600
800
1000
Time (ns)
S0
Time
Observe many single
molecule emission events!
Ensemble Emission
32 excited states
+ 32 photons
64 excited states
Time 1
hn
Time 2
Time 4
4 excited states
+ 4 photons
Time 5
etc.
Time 3
8 excited states
+ 8 photons
16 excited states
+ 16 photons
Ensemble Emission
32 excited states
+ 32 photons
64 excited states
70
60
# Excited States
Time 1
hn
Time 2
Time 4
Time 3
50
40
30
20
10
0
4 excited states
+ 4 photons
8 excited states
+ 8 photons
16 excited states
+ 16 photons
0
etc.
Emission Intensity
40
32 photons
30
16 photons
20
8 photons
10
0
1
2
3
4
5
Time
6
4
Time
Time 5
0
2
7
8
9
10
6
8
10
Excited State Decay Curve
70
*
n (t)
t / 

e
n * (0 )
# Excited States
60
50
40
30
20
10
0
0
2
4
6
8
10
Time
S1
n*(0) is the # of the excited state at time 0
n*(t) is the # of the excited state at time t
 is the lifetime of the excited state

1
=
kr + knr
Energy
Pulsed
Excitation
knr
S0
We don’t get to count the number of excited state molecules!
kr
Intensity Decay Curve
I(t)
= e-t/
I(0)
I(0) is the initial intensity at time zero
Emission Intensity
40
I(t) is the intensity at time t
10
0
1
2
3
4
5
6
7
8
9
10
Time
1
=
kr + knr
5000
4000
Intensity
 = time it takes for 63.2 % of excited states
to decay
 should always be the same for a given
molecule under the same conditions
20
0
 is the lifetime of the excited state

30
3000
2000
1000
0
0
200
400
600
Time (ns)
800
1000
Intensity Decay Curve
Linear Scale
Log Scale
1.00 -Emission
Exciting pulse
intensity
Log intensity
Emission
1/e
Exciting pulse

time
time
I(t)
I(0)
= e-t/
intensity
Spectra Decay
I(t)
I(0)
= e-t/
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
Lifetime Measurements
Sample
Source
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Frequency Domain
Time Domain
Light source
Intensity
Intensity
Light source
time
Harmonic or phase-modulation method
time
Pulsed Method
Frequency-domain Method
Measure Events with Respect to Frequency
Time
Low I0
Excitation
High I0
Excitation
Low I0
Excitation
Sample
hn
hn
hn
hn
hn
hn
I0
Frequency-domain Method
Frequency-domain Method
Excitation Modulation =
a
b
a = average intensity
b = average-to-peak intensity
Emission Modulation =
A
B
A = average intensity
B = average-to-peak intensity
(B/A)
Modulation (m) =
(b/a)
Phase Shift (f)
Frequency-domain Method
Ex Frequency ()
Modulation (m)
Phase Shift (f)
Phase (τφ) and modulation (τm) lifetimes
 f   tanf
1
 m   [(1/ m ) 1]
1
2
1/ 2
Changing , measuring m and f to calculate lifetime.
Frequency-domain Method
 m   [(1/ m ) 1]
1
2
1/ 2
 f   tanf
1
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)
Frequency-domain Method
 f   1 tanf
 m   [(1/ m ) 1]
1
2
Ex Frequency ()
Modulation (m)
Phase Shift (f)

f

m
1/ 2
Frequency-domain Instrument
Frequency-domain Method
List of Commercially Available Frequency-domain Instruments
Lifetime Measurements
Sample
Source
hn
hn
Frequency Domain
Time Domain
Light source
Intensity
Intensity
Light source
time
Harmonic or phase-modulation method
time
Pulsed Method
Time-Domain Method
Measure Events with Respect to Time
Intensity
Light source
Emission
Emission intensity is
measured following a
short excitation pulse
time
•
•
•
•
Pulsed method
Lifetimes as short as 50 fs
Multiple measurement techniques
Sources typically not as tunable as frequency domain
Time-domain Techniques
Intersystem Crossing
Excitation
Fluorescence
Phosphorescence
Internal Conversion
1 fs
1 ps
femto
pico
1 ns
nano
0.000 000 001 s
0.000 000 000 001 s
0.000 000 000 000 001 s
1 ms
1 ms
1s
micro
0.000001 s
milli
0.001 s
seconds
1s
Time-domain Techniques
TCSPC
Streak Camera
Up-conversion
1 fs
Real-time Measurement
MCS
Strobe
1 ps
1 ns
1 ms
1 ms
1s
Time-domain Techniques
1. Real-Time lifetime measurement ( > 200 ps)
2. Multi-channel scaler/photon counter ( > 1 ns)
3. Strobe –Technique ( > 250 ps)
4. Time-correlated single-photon counting ( > 20 ps)
5. Streak-camera measurements ( > 2 ps)
6. Fluorescence up-conversion ( > 150 fs)
Real-Time Lifetime
hn
Real-Time Lifetime
Source
Clock
(4)
Detector
hn
(3)
Sample
Monochromator
1) Pulsed excitation
2) Sample excitation/emission
3) Monochromator
4) Detector signal
5) Plot Signal vs. Time
(1)
(2)
Real-Time Lifetime
Detector Current
Light source
Sources
Flashlamp
Laser
Pulsed LED
Emission
time
Detector Current
Real-Time Lifetime
Instrument Response
Function (IRF)
Emission
time
•
•
•
•
Make excitation pulse width as short as possible
Time resolution is usually detector dependent
Excited-state lifetime > IRF
Lifetimes > 200 ps
Real-Time Lifetime
5000
100 averages
Intensity
4000
3000
2000
1000
0
0
200
400
600
Time (ns)
800
1000
Strobe-Technique
25 images per second
Strobe-Technique
Photon Technology International (PTI)
Strobe-Technique
Light Pulse
Measurement
Window
time
Light Pulse
Measurement
Window
time
Strobe-Technique
Light Pulse
Measurement
Window
time
Detector
Signal
time
time
Strobe-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
Strobe-Technique
(2)
(1)
(4)
(3)
1) Trigger Signal
2) Excitation Flash
(5)
3) Detector Signal Delay
4) Detect
5) Output
 > 250 ps
Time-Correlated Single-Photon Counting (TCSPC)
S1
Ex
Energy
Em
Em
Ex
Ex
S0
Time
Excited State Lifetime of an individual molecule: 0 – infinity
The sum an individual molecule lifetimes = 
Time-Correlated Single-Photon Counting (TCSPC)
Low excitation intensity:
- Low number of excited state
Time
- 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)
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
Detector Bins
Time
Time-Correlated Single-Photon Counting (TCSPC)
Sum the Photons per Bin
Detector Bins
Time
Time-Correlated Single-Photon Counting (TCSPC)
Repeat
Probability Distribution
Time-Correlated Single-Photon Counting (TCSPC)
Time-Correlated Single-Photon Counting (TCSPC)
Repeat: 10,000 counts in the peak channel
Time-Correlated Single-Photon Counting
pulsed source
2) Monochromator
(1)
3) Beam Splitter
1) to trigger PMT
exc.
monochromator
2) to sample
(2)
Start
PMT
4) Excite Sample
(3)
5) Sample emits into monochromator
6) Emission hits PMT and timer stops
7) Repeat a million times
sample
(4)
Dt
emission
monochromator
1) Pulsed excitation (10kHz)
Source:
Flash lamp
solid state LED
laser
Stop
PMT
(5)
(6)
TCSPC
1)
2)
3)
4)
5)
6)
Pulsed excitation
constant function discriminator
time-to-amplitude converter
Ex CFD triggers TAC
programmable gain amplifier
TAC voltage rises
analog-to-digital converter
Em CFD stops TAC
TAC discharges to PGA
PGA siganl to ADC for a single data point
(CFD)
(TAC)
(PGA)
(ADC)
TCSPC
48
TCSPC
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
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
Resolution vs. Acquisition Time
Detector Bins
Time
Detector Bins
Time
5 ns wide bin = 5 ns resolution
10 minutes to acquire 10,000 counts
1 ns wide bin = 1 ns resolution
50 minutes to acquire 10,000 counts
Resolution
Acquisition Time
Resolution
Acquisition Time
Repetition Rate to High
hn
hn
Real
start-stop-time
Time
Signal
Repetition Rate to High
time
If the rep rate is too high the
histogram is biased to shorter times!
Measured  < Real 
Keep rep rate at least 10 times slower than your 
Intensity to High
Single 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.
Side Note: PMT Lifetime
Photoelectric Effect
Photon Energy - binding energy = electron kinetic energy
Side Note: PMT Lifetime
Photoelectric Effect
Photon Energy - binding energy = electron kinetic energy
Higher Energy Photons = Faster Signal
Measured Lifetime < Real Lifetime
Streak-Camera
Temporal profile from Spatial profile
Laser Pointer Duty Cycle
Distance
Calculating Duty Cycle
Length
(spatial)
Pointer Motion
m/s
Use length to calculate time
Streak-Camera
Cathode Ray Tube
+
e-
Streak-Camera
(1)
(4)
(2)
Source
(3)
hn
Sample
Monochromator
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
Streak-Camera
5000
Intensity
4000
Calculating Duty Cycle
3000
2000
1000
0
0
200
400
600
800
1000
Time (ns)
Distance
Intensity
Length
(spatial)
Length
time(0)
Pointer Motion
m/s
Use length to calculate time
-
time(t)
+
Sweep Rate
m/s
eUse length and intensity to calculate lifetime
Streak-Camera
(1)
(4)
(2)
Source
(3)
hn
Sample
Monochromator
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
Streak-Camera
Electrons that arrive first hit the detector at a different
position compared to electrons that arrive later.
Streak-Camera
Streak-Camera
http://www.youtube.com/watch?v=rA6A7haKFwI
Streak-Camera
• 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
Instrument Response Functions
TCSPC
Time resolution down to 2ps or even 100s of femtoseconds.
Fluorescence up-conversion
Sum Frequency Method
ωsum = ω1 + ω2
Fluorescence up-conversion
excitation beam
gate beam
(1)
(1)
(2)
(3)
(4)
(5)
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
(6)
Fluorescence up-conversion
Graph of td vs intensity
Intensity
Intensity
Excitation pulse
Emission
time
time
Intensity
Intensity
Excitation Gate
pulse
pulse
td1
Summed Light
at time 1
time
time
Gate
pulse
Intensity
Intensity
Excitation
pulse
td2
time
Control td and measure only summed light
Summed Light
at time 2
time
Fluorescence up-conversion
excitation beam
gate beam
(1)
(1)
(2)
(3)
(4)
(5)
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
(6)
Signal is only measured when gate is pulsed
td is controlled by the delay track
Light Travels 0.9 m in 1 ns
Comparison
Sum Frequency Generation
TCSPC
Intensity
Intensity
Detector Bins
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)
time
Control excitation measure td
Limited by detector response.
Data point limited by PMT (10 ps)
Fluorescence up-conversion
Fluorescence up-conversion
Phys . Chem. Chem. Phys. 2005, 7, 1716 – 1725.
Fluorescence up-conversion
Fluorescence up-conversion
• 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
74
Decay Fitting
Non-exponential decay
Exponential decay
I(t)
I(0)
= e-t/
Non-exponential Decay (Log)
Non-exponential decay
Intensity
Intensity
Exponential decay
Time
I(t)
I(0)
Time
= e-t/
Non-exponential
Possible explanations:
- Two or more emitters
- In homogeneous samples (QDs)
- Dual Emission
- Multiple emissive sites
On surfaces
Polymer Films
Peptides
Dual Emission
Non-exponential Decay
Linear Scale
Intensity
Biexponential Fit
I(t)
= A1e-t/1 + A2e-t/2
I(0)
t/ns
Log I
Log Scale
50 ns
5 ns
t/ns
A1
1
A2
2
= amplitude of component 1
= lifetime of component 1
= amplitude of component 2
= lifetime of component 2
Non-exponential Decay
I(t)
= A1e-t/1 + A2e-t/2
I(0)
Limitations of Multi-exponential Fits
Biexponential Fits
Linear Scale: No difference
Log Scale: minor differences at 30–50 ns
1 = 5.5 ns and 2 = 8.0 ns
or
1 = 4.5 ns and 2 = 6.7 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.
Fitting Data
Multi-exponential Fits
Exponential
The Data
y = A1e-k1t
c2 = 26.466
Bi-exponential
Tri-exponential
c2 = 2.133
y = A1e-k1t + Ae-k2t
c2 = 1.194
y = A1e-k1t + Ae-k2t + A3e-k3t
It could be worse!
J. of Political Economy
2005, 113, 949
Time-resolved Emission End
Any Questions?

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