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1 Ken Hanson MWF 9:00 – 9:50 am Office Hours MWF 10:00-11:00 CHM 5175: Part 2.6 Time-resolved emission Source h Sample Detector Clock.

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Presentation on theme: "1 Ken Hanson MWF 9:00 – 9:50 am Office Hours MWF 10:00-11:00 CHM 5175: Part 2.6 Time-resolved emission Source h Sample Detector Clock."— Presentation transcript:

1 1 Ken Hanson MWF 9:00 – 9:50 am Office Hours MWF 10:00-11:00 CHM 5175: Part 2.6 Time-resolved emission Source h Sample Detector Clock

2 Steady-state Emission Intensity vs. Wavelength Source Sample h Information about emission intensity (yield) and wavelength. S0S0 S1S1 Energy Constant Excitation Constant Emission Equilibrium between absorption, non-emissive decay and emission. Non- emissive decay h

3 Time-resolved Emission Intensity vs. Time Information about emission lifetimes. S0S0 S1S1 Energy Pulsed Excitation krkr k nr Short Burst of Light Competition between non-emissive decay and emissive rates. Source Sample h h

4 Single Molecule Emission Excited State Lifetime of an individual molecule: 0 – infinity Anthracene Excited state Lifetime: Time spent in the excited state (S 1 ) prior to radiative (k r ) or non- radiative decay. (k r ) Ex Em S0S0 S1S1 Energy Time Ex Em Ex

5 Ensemble Emission Time-resolved Emission Intensity vs. Time Single Molecule Emission Excited State Lifetime of an individual molecule: 0 – infinity Observe many single molecule emission events! Ex Em S0S0 S1S1 Energy Time Ex Em Ex

6 Ensemble Emission h Time 1 32 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.

7 Ensemble Emission 32 photons 16 photons 8 photons

8 k r + k nr Excited State Decay Curve 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 S0S0 S1S1 Energy Pulsed Excitation krkr k nr = 1 We dont get to count the number of excited state molecules!

9 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 state = e -t/ k r + k nr = 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 I(t) I(0)

10 time intensity 1.00 -- 1/e Exciting pulse Emission time Log intensity Exciting pulse Emission Intensity Decay Curve Linear ScaleLog Scale = e -t/ I(t) I(0)

11 Spectra Decay = e -t/ I(t) I(0) intensity

12 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 k r and k nr (if you know ) GFP- Nobel prize, expression studies Sensing

13 Lifetime Measurements Intensity time Light source Time Domain Pulsed Method Harmonic or phase-modulation method Frequency Domain time Intensity Light source Source Sample h h

14 h h h h Low I 0 Excitation High I 0 Excitation h h Low I 0 Excitation Time Frequency-domain Method I0I0 Measure Events with Respect to Frequency

15 Frequency-domain Method

16 Excitation Modulation = a b a = average intensity b = average-to-peak intensity Emission Modulation = A B A = average intensity B = average-to-peak intensity Modulation (m) = (B/A) (b/a) Phase Shift ( )

17 Frequency-domain Method Modulation (m) Phase Shift ( ) Ex Frequency ( ) Changing, measuring m and to calculate lifetime. Phase (τ φ ) and modulation (τ m ) lifetimes

18 Frequency-domain Method

19 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)

20 Frequency-domain Method m Modulation (m) Phase Shift ( ) Ex Frequency ( )

21 Frequency-domain Instrument

22 Frequency-domain Method List of Commercially Available Frequency-domain Instruments

23 Lifetime Measurements Intensity time Light source Time Domain Pulsed Method Harmonic or phase-modulation method Frequency Domain time Intensity Light source Source Sample h h

24 Intensity 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

25 Time-domain Techniques 1 s 1 ms 1 s 1 ns1 ps1 fs seconds milli micronano pico femto 0.001 s0.000001 s0.000 000 001 s 0.000 000 000 001 s 0.000 000 000 000 001 s 1 s Excitation Phosphorescence Fluorescence Internal Conversion Intersystem Crossing

26 TCSPC Time-domain Techniques 1 s 1 ms 1 s 1 ns1 ps1 fs Streak Camera MCS Strobe Up-conversion Real-time Measurement

27 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)

28 Real-Time Lifetime h

29 Source h 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

30 Real-Time Lifetime Light source Detector Current time Emission Sources Flashlamp Laser Pulsed LED

31 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) Detector Current time Emission

32 Real-Time Lifetime 100 averages

33 Strobe-Technique 25 images per second

34 Strobe-Technique Photon Technology International (PTI)

35 Strobe-Technique time Light Pulse Measurement Window time Light Pulse Measurement Window

36 Strobe-Technique time Light Pulse Measurement Window time Detector Signal

37 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

38 (1) (2) (3) (4) (5) Strobe-Technique 1) Trigger Signal 2) Excitation Flash 3) Detector Signal Delay 4) Detect 5) Output > 250 ps

39 Time-Correlated Single-Photon Counting (TCSPC) Excited State Lifetime of an individual molecule: 0 – infinity The sum an individual molecule lifetimes = Ex Em S0S0 S1S1 Energy Time Ex Em Ex

40 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

41 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 t after pulse at t = 0 Time Detector Bins Time-Correlated Single-Photon Counting (TCSPC)

42 Time Detector Bins Sum the Photons per Bin

43 Time-Correlated Single-Photon Counting (TCSPC) Probability Distribution Repeat

44 Time-Correlated Single-Photon Counting (TCSPC)

45 Repeat: 10,000 counts in the peak channel Time-Correlated Single-Photon Counting (TCSPC)

46 Time-Correlated Single-Photon Counting Source: Flash lamp solid state LED laser Start PMT Stop PMT sample exc. monochromator emission monochromator 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)

47 constant function discriminator (CFD) time-to-amplitude converter (TAC) programmable gain amplifier (PGA) analog-to-digital converter (ADC) TCSPC 1) Pulsed excitation 2) Ex CFD triggers TAC 3) TAC voltage rises 4) Em CFD stops TAC 5) TAC discharges to PGA 6) PGA siganl to ADC for a single data point

48 48 TCSPC

49 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 s – 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

50 Acquisition Time Time Detector Bins Resolution vs. Acquisition Time 5 ns wide bin = 5 ns resolution 10 minutes to acquire 10,000 counts Time Detector Bins 1 ns wide bin = 1 ns resolution 50 minutes to acquire 10,000 counts Resolution Acquisition Time Resolution

51 Time Repetition Rate to High h h Real start-stop-time

52 Signal 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

53 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!

54 Photoelectric Effect Photon Energy - binding energy = electron kinetic energy Side Note: PMT Lifetime

55 Photoelectric Effect Photon Energy - binding energy = electron kinetic energy Higher Energy Photons = Faster Signal Measured Lifetime < Real Lifetime

56 Streak-Camera Temporal profile from Spatial profile Laser Pointer Duty Cycle Calculating Duty Cycle Pointer Motion m/s Distance Length (spatial) Use length to calculate time

57 Streak-Camera Cathode Ray Tube e-e- + -

58 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 h Sample Monochromator (3) (4)

59 Calculating Duty Cycle Pointer Motion m/s Distance Length (spatial) Use length to calculate time Streak-Camera Sweep Rate m/s Length Use length and intensity to calculate lifetime e-e- time(0) time(t) + - Intensity

60 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 h Sample Monochromator (3) (4)

61 Electrons that arrive first hit the detector at a different position compared to electrons that arrive later. Streak-Camera

62

63 http://www.youtube.com/watch?v=rA6A7haKFwI Streak-Camera

64 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

65 Time resolution down to 2ps or even 100s of femtoseconds. TCSPC Instrument Response Functions

66 Fluorescence up-conversion Sum Frequency Method ω sum = ω 1 + ω 2

67 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 beam gate beam (1) (3) (5) (6)

68 Fluorescence up-conversion Excitation pulse Emission Intensity Excitation pulse Intensity Gate pulse t d1 Intensity Summed Light at time 1 time Excitation pulse Intensity Gate pulse t d2 Intensity Summed Light at time 2 time Intensity time Control t d and measure only summed light Graph of t d vs intensity

69 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 beam gate beam (1) (3) (5) (6) Signal is only measured when gate is pulsed t d is controlled by the delay track Light Travels 0.9 m in 1 ns

70 Comparison Intensity time Control t d 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 Intensity time Limited by detector response. Data point limited by PMT (10 ps) Control excitation measure t d

71 Fluorescence up-conversion

72 Phys. Chem. Chem. Phys. 2005, 7, 1716 – 1725. Fluorescence up-conversion

73

74 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

75 Decay Fitting Exponential decay = e -t/ I(t) I(0) Non-exponential decay

76 Non-exponential Decay (Log) Exponential decay Non-exponential decay Time Intensity = e -t/ I(t) I(0) Intensity

77 Non-exponential Possible explanations: - Two or more emitters - In homogeneous samples (QDs) - Dual Emission - Multiple emissive sites On surfaces Polymer Films Peptides Dual Emission

78 5 ns 50 ns Non-exponential Decay = A 1 e -t/ + A 2 e -t/ I(t) I(0) A 1 = amplitude of component 1 1 = lifetime of component 1 A 2 = amplitude of component 2 2 = lifetime of component 2 Linear Scale Log Scale Biexponential Fit

79 Non-exponential Decay = A 1 e -t/ + A 2 e -t/ I(t) I(0)

80 Limitations of Multi-exponential Fits Linear Scale: No difference Log 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 ns or 1 = 4.5 ns and 2 = 6.7 ns

81 Fitting Data

82 y = A 1 e -k 1 t + A­e -k 2 t + A 3 e -k 3 t 2 = 26.466 y = A 1 e -k 1 t + A­e -k 2 t 2 = 2.133 The Data Exponential Bi-exponential Tri-exponential 2 = 1.194 Multi-exponential Fits y = A 1 e -k 1 t

83 J. of Political Economy 2005, 113, 949 It could be worse!

84 Time-resolved Emission End Any Questions?


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