chm 5175: part 2.6

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

Steady-state Emission

550 600 650 700 750 800 8500

50000

100000

150000

200000

250000

Inte

nsity

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

Time-resolved Emission

Intensity vs. Time

Information about emission lifetimes.

S0

S1

EnergyPulsed

Excitationkrknr 0 200 400 600 800 1000

0

1000

2000

3000

4000

5000

Inte

nsity

Time (ns)

Short Burst of Light

Competition between non-emissive decay and emissive rates.

Source Samplehn

hn

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

Ensemble Emission

Time-resolved EmissionIntensity vs. Time

0 200 400 600 800 10000

1000

2000

3000

4000

5000

Inte

nsity

Time (ns)

Single Molecule Emission


Observe many single molecule emission events!

Ex Em

S0

S1

Energy

Time

Ex Em Ex

Ensemble Emission

hn Time 1

64 excited states32 excited states

+ 32 photons

Time 2

Time 3

16 excited states + 16 photons

Time 4



Time 5

etc.

Ensemble Emission

hn Time 1

64 excited states32 excited states

+ 32 photons

Time 2

Time 3


Time 4



Time 5

etc.

0 2 4 6 8 100

10

20

30

40

50

60

70

# Ex

cite

d St

ates

Time

0 1 2 3 4 5 6 7 8 9 100

10

20

30

40

Emis

sion

Inte

nsity

Time

32 photons

16 photons

8 photons

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

# Ex

cite

d St

ates

Time

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

S0

S1

EnergyPulsed

Excitationkrknr

=1

We don’t get to count the number of excited state molecules!

Intensity Decay Curve

I(0) is the initial intensity at time zero

I(t) is the intensity at time t

is the lifetime of the excited state0 1 2 3 4 5 6 7 8 9 10

0

10

20

30

40

Emis

sion

Inte

nsity

Time

= e-t/

kr + knr =

1

= time it takes for 63.2 % of excited states to decay

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

nsity

Time (ns)

I(t)I(0)

time

intensity

1.00 --

1/e

Exciting pulse

Emission

time

Log intensity

Exciting pulse

Emission

Intensity Decay Curve

Linear Scale Log Scale

= e-t/I(t)I(0)

Spectra Decay

= e-t/I(t)I(0)

inte

nsity

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

Inte

nsity

time

Light source

Time Domain

Pulsed MethodHarmonic or phase-modulation method

Frequency Domain

time

Inte

nsity

Light source

Source Samplehn

hn

Samplehn

hn

hnhn

Low I0 Excitation

High I0 Excitation

hnhn

Low I0 Excitation

Time

Frequency-domain Method

I0

Measure Events with Respect to Frequency


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)


Modulation (m)

Phase Shift (f)

Ex Frequency ()

2/121 ]1)/1[( mm

ff tan1

Changing , measuring m and f to calculate lifetime.

Phase (τφ) and modulation (τm) lifetimes

ff tan12/121 ]1)/1[( mm



• 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)


2/121 ]1)/1[( mm

ff tan1

f

m

Modulation (m)

Phase Shift (f)

Ex Frequency ()

Frequency-domain Instrument


List of Commercially Available Frequency-domain Instruments

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

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

Time-domain Techniques

1 s1 ms1 ms1 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

TCSPC


1 s1 ms1 ms1 ns1 ps1 fs

Streak Camera MCSStrobeUp-conversion

Real-time Measurement


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

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

Real-Time Lifetime

Light source De

tect

or C

urre

nt

time

Emission

SourcesFlashlampLaserPulsed LED

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)

Dete

ctor

Cur

rent

time

Emission

Real-Time Lifetime

0 200 400 600 800 10000

1000

2000

3000

4000

5000

Inte

nsity

Time (ns)

100 averages

Strobe-Technique

25 images per second

Strobe-Technique

Photon Technology International (PTI)

Strobe-Technique

time

Light PulseMeasurement Window

time

Light PulseMeasurement Window

Strobe-Technique

time

Light Pulse

Measurement Window

time

time

Dete

ctor

Si

gnal

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

(1) (2)

(3) (4)

(5)

Strobe-Technique

1) Trigger Signal

2) Excitation Flash

3) Detector Signal Delay

4) Detect

5) Output > 250 ps

Time-Correlated Single-Photon Counting (TCSPC)


The sum an individual molecule lifetimes =

Ex Em

S0

S1

Energy

Time

Ex Em Ex

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

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

Detector Bins

Sum the Photons per Bin


Probability Distribution

Repeat

Excitation Pulse


Repeat: 10,000 counts in the peak channel


Time-Correlated Single-Photon CountingSource:

Flash lampsolid state LED laser

Start PMT

Stop PMTsample

exc. monochromator

emis

sion

m

onoc

hrom

ator

pulsed source

Dt

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)

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

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

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

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

Time

Repetition Rate to High

hnhn

Real start-stop-time

Sign

al

time

Repetition Rate to High

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

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!

Photoelectric Effect

Photon Energy - binding energy = electron kinetic energy

Side Note: PMT Lifetime

Side Note: PMT LifetimePhotoelectric Effect

Photon Energy - binding energy = electron kinetic energy

Higher Energy Photons = Faster Signal

Measured Lifetime < Real Lifetime

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

Streak-CameraCathode Ray Tube

e-

+

-

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


(3)

(4)

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

nsity

Time (ns)

+-

Intensity

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


(3)

(4)

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

http://www.youtube.com/watch?v=rA6A7haKFwI

• 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

Streak-Camera

Time resolution down to 2ps or even 100s of femtoseconds.

TCSPC

Instrument Response Functions

Fluorescence up-conversion

Sum Frequency Method

ωsum = ω1 + ω2


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)

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


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

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

Phys . Chem. Chem. Phys. 2005, 7, 1716 – 1725.


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


Decay Fitting

Exponential decay

= e-t/I(t)I(0)

Non-exponential decay

Non-exponential Decay (Log)

Exponential decay Non-exponential decay

Time Time

Inte

nsity

= e-t/I(t)I(0)

Inte

nsity

Non-exponential

Possible explanations:- Two or more emitters

- In homogeneous samples (QDs)

- Dual Emission

- Multiple emissive sites

On surfaces

Polymer Films

Peptides

Dual Emission

Intensity

t / ns

Log

I

t / ns

5 ns50 ns

Non-exponential Decay

= A1e-t/1 + A2e-t/2I(t)I(0)

A1 = amplitude of component 11 = lifetime of component 1A2 = amplitude of component 22 = lifetime of component 2

Linear Scale

Log Scale

Biexponential Fit

Non-exponential Decay

= A1e-t/1 + A2e-t/2I(t)I(0)

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

1 = 5.5 ns and 2 = 8.0 nsor

1 = 4.5 ns and 2 = 6.7 ns

Fitting Data

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

J. of Political Economy 2005, 113, 949

It could be worse!

Time-resolved Emission End

Any Questions?

chm 5175: part 2.6

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