Time and Frequency Domain
Lifetime Measurements
Fluoro’Fest 2009
Jim Mattheis, Ph.D.
June 4, 2009
© 2009 HORIBA, Ltd. All rights reserved.
Overview
„What is a fluorescence lifetime?
„Why measure fluorescence lifetimes
„Two approaches to lifetimes
zFrequency-Domain
zTime-Domain by TCSPC
– Time Correlated Single Photon Counting
„Selecting your method
„Applications
© 2009 HORIBA, Ltd. All rights reserved.
What is the fluorescence lifetime?
Internal Conversion
Absorption
femtoseconds
Blue Excitation
S2 excited state
S1 excited state
Fluorescence
nanoseconds
Ground State
Electrons
© 2009 HORIBA, Ltd. All rights reserved.
Nonradiative
dissipation
1.5
M
=
)
(
)
0.5
0
Lifetime By
Frequency Domain
-0.5
-1
φ
-1.5
φ
TIME
90
80
70
60
50
40
30
20
10
0
1
© 2009 HORIBA, Ltd. All rights reserved.
10
100
Frequency, MHz
τφ = tan(φ)/ω
Described By
2
0.5
τM = (1/M
phase -1)
shift /ω
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1000
modulation
M
AMPLITUDE
1
(
What is the fluorescence lifetime?
1
I(t)
0.8
0.6
τ=1/e=37%
0.4
0.2
0
0
Population of Molecules Excited
With Instantaneous Flash
© 2009 HORIBA, Ltd. All rights reserved.
500
time, ps
1000
Why Measure Lifetimes?
„Independent of sample intensity
z Absolute measurement
z Concentration independent
„Sensitive to local environment
z pH, local charged group, quenchers
z Temperature, polarity, viscosity
„More information
z Additional dimension to fluorescence data map
z Increases specificity of measurement
© 2009 HORIBA, Ltd. All rights reserved.
Why fluorescence lifetimes
„More information
zEnergy transfer
zCollisional quenching
zRotational diffusion
zExcited state events
– Electron / proton transfer
© 2009 HORIBA, Ltd. All rights reserved.
PRINCIPLES OF TCSPC
© 2009 HORIBA, Ltd. All rights reserved.
Principle of TCSPC
„ Based on the fact that the probability of
detecting a single photon at time t after an
exciting pulse is proportional to the
fluorescence intensity at that time
„ Fluorescence decay is reconstructed after
timing and recording the single photon
following a large number of exciting pulses
„ Mathematical fitting to models of fluorescence
lifetime(s)
© 2009 HORIBA, Ltd. All rights reserved.
Principles of TCSPC
„Experimental observable is intensity
„Pulsed light source
„Photon detector – e.g. photomultiplier
„Time to amplitude converter - TAC
„Multichannel analyzer - ADC/MCA
zKeep track of all the timed photon events
zHistogram representing the decay
„Mathematical fitting of the histogram to
lifetime models
© 2009 HORIBA, Ltd. All rights reserved.
TBX-04
PHOTON COUNTS
12000
10000
Cumulative
histogram
8000
6000
4000
0
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
statistical
single photon
events
Em.
filter
2000
NanoLed
TIME, CHANNELS
S
HJY-IBH FluoroHub-B
periodic
pulses
α ≤ 2%
TIME
DIGITISER
COMPUTER
TemPro TCSPC Instrument Principle
© 2009 HORIBA, Ltd. All rights reserved.
100000
PHOTON COUNTS
Time Domain
Convolution
Principle
δ−pulse
decay
10000
1000
100
Intensity as
function of time:
I(t)=α exp (-t/τ)
10
1
-10
-5
0
TIME, CHANNELS
100000
δ−1
PHOTON COUNTS
10000
δ−2
1000
δ0
5
10
convolved
decay
δ−3
100
10
δ−4
1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
TIME, CHANNELS
© 2009 HORIBA, Ltd. All rights reserved.
Lamp intensity
as function of time:
L(t)
Fluorescence Convolution:
F(t)= I(t) ⊗ L(t)
TCSPC Lifetime of HSA
Human Serum Albumin
2.5mg/ml
295 nm NanoLED source
Lifetimes ns (fraction)
0.45 +/-0.26 (2%)
3.95 +/-0.27 (38%)
7.25 +/-0.08 (60%)
© 2009 HORIBA, Ltd. All rights reserved.
ChiR2 = 1.08
PRINCIPLES OF FREQUENCY
DOMAIN
© 2009 HORIBA, Ltd. All rights reserved.
Principles of Frequency-Domain
„Based on the response of a population
of fluorophores to sinusoidally, intensity
modulated excitation light
„Emission is modulated at the same
frequency but phase shifted and demodulated
„A frequency response curve is
generated
„Mathematical fitting to lifetime models
© 2009 HORIBA, Ltd. All rights reserved.
Principles of Frequency Domain
„Two observables
zPhase shift
zModulation ratio
„Light source (intensity modulated)
„Two synthesizers
„Two amplifiers
„PMT
© 2009 HORIBA, Ltd. All rights reserved.
φ
M
Sin & Cos
Transforms
Spectracq
Rf+∆f
Rf
X
MHz
MASTER Rf
450W
cw xenon
amp
SLAVE Rf + ∆f
Cross correlation
R928P
PMT
amp
filter
Pockels Cell
sample turret
© 2009 HORIBA, Ltd. All rights reserved.
The Newest Approach to Frequency-Domain
„8 frequencies in parallel
4
„10 increase in acquisition rates
– 1ms measurement cycle
„103 increase in temporal range
– 10 ms through 10 ps
– 500Hz through 320MHz
© 2009 HORIBA, Ltd. All rights reserved.
Multiple Frequencies Based System
Five (5) Frequencies
Three (3) Frequencies
Time (ms) reveals phase and demodulation
Time (ms)
Transform
values
0
© 2009 HORIBA, Ltd. All rights reserved.
64
Frequency (Hz)
12
0
82.5
Frequency (Hz)
1
New Approach - 8 Simultaneous Frequencies
Relative Intensity
Sum of Eight Frequencies
Time - ms
© 2009 HORIBA, Ltd. All rights reserved.
80
Phase shift
70
60
50
40
30
20
10
0
10
100
MHz
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1000
Demodulation
90
Principle of MF2
To PMT
PMT
To
LED
LED
T-box sample optics
Electronics
© 2009 HORIBA, Ltd. All rights reserved.
Bi-Exponential POP+9CA (MeOH)
Experiment duration < 1s
© 2009 HORIBA, Ltd. All rights reserved.
Quantum Dot Lifetime
MF2 Frequency Stitching
© 2009 HORIBA, Ltd. All rights reserved.
Time-Domain or Frequency-Domain
SELECT YOUR METHOD
© 2009 HORIBA, Ltd. All rights reserved.
Select Your Method
Phase & Modulation
1
90 Ο
Ο Ο
+ Ο
+ Ο
+ Ο
+ Ο
+ Ο
+ Ο
+ +
+
80
7ns
Phase Angle (degrees)
0.1
2 + 7 ns
0.01
0.001
2ns
0.0001
-5
0
5
10
15
Time (nanoseconds)
20
Ο
Ο
+
+
20
+
Ο
+
+
Ο Ο
+
Ο
Ο
Ο
Ο
Ο
Ο
Ο
Ο
1.00E+7
Frequency (Log Plot)
0.6
2ns
Ο
Ο
+
0.7
Ο
0.5
Ο
Ο
+
+
Ο
+ Ο
2 + 7 ns
+
+
30
1
+
+
0.9
+ +
+
Ο
0.8
+
Ο
+
7ns
40
Ο
Ο
+
50
+
+ Ο
+ Ο
+
+
Ο
Ο
Ο
0
1.00E+6
Ο
+
60
Same phenomenon
© 2009 HORIBA, Ltd. All rights reserved.
+
70
10
0.00001
Ο
0.4
Ο
+
Ο
+
1.00E+8
+
0.3
0.2
+ + 0.1
0
3.00E+8
Same results
Modulation
TCSPC
Applications
ofof
Frequency
Applications
TCSPC
„Multi-component analysis (sum of exp.)
„Non-exponential decays
„Anisotropy decay
„FRET
„Quenching
„TRES
„Solvent / Spectral relaxation
Same phenomenon
© 2009 HORIBA, Ltd. All rights reserved.
Same results
Frequency and Time domain comparison
Advantages
Disadvantages
¾
¾
¾
Time
Domain
TCSPC
¾
¾
¾
¾
Single-photon sensitivity works well with weak
samples; <1nM routine with laser excitation
Wide temporal range (10ps to seconds) depending
on excitation source and detector combination
Intuitive data interpretation, uses Poisson statistics
Rapid data acquisition with diode excitation
sources (especially complex decays)
Scatter can be fitted as additional component
Compatible with Ti-sapphire laser systems
¾
¾
¾
¾
Time consuming measurement if the emission
intensity of the sample is low
Multiple excitation sources may be required
Xenon illuminator lamp can’t be used for timedomain excitation
Shortest measurable lifetime depends on instrument
response - varies depending on detector and
excitation source combination
Pulse “pile-up” can limit data acquisition rate with
high quantum yield samples – need to monitor count
rate
¾
Freq
Domain
MFF
Fast data collection, especially for anisotropy
decay
¾ High accuracy of rf sinusoidal modulation
¾ Free from convolution
¾ short (20ps) lifetime resolved with simple
apparatus
¾ Use any CW source
Xenon or Hg, Hg(Xe) lamps
Relatively low cost laser (HeCd or Ar+ are common)
¾ Pulse broadening does not affect temporal
resolution in frequency domain
=> Can use double-grating monochromators easily
© 2009 HORIBA, Ltd. All rights reserved.
¾
¾
Need to measure against a standard
No visualization of the decay curve
Advantages of TCSPC
„Sensitivity – photon counting
zWorks well with weak samples
z<1nM with laser excitation
„Solid statistical basis – Poisson distribution
„Visually intuitive decay curve
zIntensity versus time
zCareful of visual fitting!
„Wavelengths into the NIR
© 2009 HORIBA, Ltd. All rights reserved.
Advantages of Frequency-Domain
„Short lifetimes with base instruments
z 10 ps with Xenon lamp
„Two experimental observables
z Phase
z Modulation
„Faster measurements
z Lifetime / Anisotropy Kinetics (1 ms)
„Handles wide differences in lifetimes
z Possible 10 ms – 10 ps in one experiment
„Use any cw light source
© 2009 HORIBA, Ltd. All rights reserved.
Disadvantages of TCSPC
„Pulse pile-up can limit data acquisition rate
– lose Poisson statistics, bias results
„Tunable light sources not as available (low
performance or high costs)
„ Must reconfigure instrument for short
lifetimes – MCP, electronics, light sources
„Dark count limited– longer lifetimes, NIR
© 2009 HORIBA, Ltd. All rights reserved.
Disadvantages of Frequency-Domain
„Analog acquisition lower sensitivity at
limits of detectibility
„Frequency space visually less intuitive
zPhase & Modulation vs log(frequency)
„Error estimates not based on Poisson
distribution
„More complexity in removing
background signal
© 2009 HORIBA, Ltd. All rights reserved.
TIME & FREQUENCY APPLICATIONS
© 2009 HORIBA, Ltd. All rights reserved.
Sample 1 (B2mApo) - Double Exponential Decay
Ex 277 nanoled, em 350 mono
1.816ns (66.0%)
4.149ns (34.0%)
ChisqrR = 1.16
T1 = 64.24338
ch;
1.816003E-09 sec
S.Dev = 1.541728E-11 sec
T2 = 146.7808
ch;
4.149133E-09 sec
S.Dev = 1.982471E-11 sec
A = 0.8738365
S.Dev = 9.938859E-02
B1 = 3.084404E-02 [ 66.01 Rel.Ampl]
S.Dev = 6.169639E-05
B2 = 6.95154E-03 [ 33.99 Rel.Ampl]
S.Dev = 2.528393E-05
© 2009 HORIBA, Ltd. All rights reserved.
Sample 4 (alexa488) - Nanoled 450nm, em 530nm
T1 = 145.6771
ch;4.117936E-09 sec
S.Dev = 5.887733E-12 sec
A = 1.285427
S.Dev = 0.1392763
B1 = 4712.9
[ 100.00 Rel.Ampl]
S.Dev = 5.771924
CHISQ = 1.037199
[ 1111 degrees of freedom ]
© 2009 HORIBA, Ltd. All rights reserved.
Sample 4 (alexa488) vs. Ludox
Ex 475 moduled LED+475bp, em 500lp
Tau1=4.138ns (0.935) Tau2=0.369ns (0.0647) Chi2R = 0.795
© 2009 HORIBA, Ltd. All rights reserved.
GFP: the Green Fluorescence Protein
biologically interesting – naturally fluorescent
0.734ns (13%)
3.027ns (84%)
11.04ns (2.7%)
© 2009 HORIBA, Ltd. All rights reserved.
Hematoporphyrin in buffer (pH 7.4)
15.12ns (95%)
5.5ns (4.5%)
0.61ns (0.5%)
© 2009 HORIBA, Ltd. All rights reserved.
Raw Phase Data: 20 ms/pt
Stopped-Flow Experiment
9-CA 11.8ns
Bi-exponential
11.8 & 4.0 ns
© 2009 HORIBA, Ltd. All rights reserved.
Mix Anthracene
4ns
Time Resolution: 20 ms
© 2009 HORIBA, Ltd. All rights reserved.
Best Tool For The Job
„ TCSPC
z Sensitivity
z Poisson statistics
z Measure into NIR
z Intuitive decay display
© 2009 HORIBA, Ltd. All rights reserved.
„ Frequency-Domain
z 10 ps lifetimes
z Fast experiments
z Wide time range
z CW excitation sources
Thank you
© 2009 HORIBA, Ltd. All rights reserved.
© 2009 HORIBA, Ltd. All rights reserved.