Transcript HORIBA Jobin Yvon Fluorescence Division Presents: Time

HORIBA Jobin Yvon Fluorescence
Division Presents:
Time-Resolved Fluorescence
Spectroscopy
Edison, NJ
March 15, 2005
Fluorescence: a type of light emission
• First observed from quinine by Sir J. F. W. Herschel
in 1845
Yellow glass of wine
Em filter > 400 nm
1853 G.G. Stoke
coined term
“fluorescence”
Blue glass
Filter
Church Window!
<400nm
Quinine
Solution
Common Fluorophores
Typically, Aromatic molecules
– Quinine, ex 350/em 450
– Fluorescein, ex 485/520
– Rhodamine B, ex 550/570
– POPOP, ex 360/em 420
– Coumarin, ex 350/em 450
– Acridine Orange, ex 330/em 500
Common Fluorophores
Absorption
femtoseconds
Internal Conversion
Absorbance energy
Blue Excitation
S2 excited state
S1 excited state
Fluorescence
nanoseconds
Ground State
Electrons
Nonradiative
dissipation
Basic Principles of Fluorescence
• Emission at longer wavelength than
excitation (Stoke shift)
• Emission spectrum does not vary with
excitation wavelength
• Excitation spectrum same as abs
spectrum
• Emission spectrum is a mirror image of its
excitation/abs spectrum
“Stokes” shift
Absorption vs Emission
E = hc / 

Time Resolved Fluorescence
• What’s happening during the time of the
fluorescence emission
• Fluorescence Lifetime
What is a Fluorescence Lifetime?
Random Decay Back to Ground State:
Each Molecule Emits 1 Photon
1
I(t)
0.8
0.6
t=1/e=37%
0.4
0.2
0
0
Population of Molecules Excited
With Instantaneous Flash
500
time, ps
1000
Why Measure Lifetimes?
•
•
•
•
Absolute measurement - lifetime normally independent
of sample concentration
Lifetime can be used as probe of local environment
(e.g. polarity, pH, temperature etc)
Additional dimension to fluorescence data map increases measurement specificity
Dynamic vs static – e.g. measure rotational correlation
times and energy transfer using lifetime data
Time Domain
TCSPC
Time Correlated Single Photon Counting
TBX-04
PHOTON COUNTS
PHOTON COUNTS
12000 12000
10000 10000
8000 8000
6000 6000
Cumulative
histogram
4000 4000
2000 2000
0
0
36
35
34
33
32
36
31
35
30
34
29
33
28
32
27
31
26
30
25
29
24
28
23
27
22
26
21
25
20
24
19
23
18
22
17
21
16
20
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14
18
13
17
12
16
11
15
10
14
913
812
711
610
59
48
37
26
15
4
3
2
1
statistical
single photon
events
TIME,
CHANNELS
TIME,
CHANNELS
nanoled
S
periodic pulses
CFD
SYNC
 ≤ 2%
TAC rate 1MHz
Coaxial Delay 50 Ns
Sync delay 20 ns
MCA
IBH 5000U
V
TAC
TCSPC Instrument Principle
100000
PHOTON
PHOTONCOUNTS
COUNTS
10000
Time Domain
Convolution
Principle
d-pulse
decay
1000
100
Intensity as
function of time:
I(t)= exp (-t/t)
10
1
-10
-10
-5
-5
0
0
TIME, CHANNELS
TIME, CHANNELS
100000
100000
d-1
PHOTON
PHOTON COUNTS
COUNTS
10000
d-2
1000
100
10
d0
5
5
10
10
convolved
decay
d-3
d-4
1
0 1
10
11
12
13
14
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19
20
1 22 33 44 55 66 77 88 99 10
1011
1112
1213
1314
1415
1516
1617
1718
1819
1920
20
TIME,
CHANNELS
TIME,
TIME, CHANNELS
CHANNELS
Lamp intensity
as function of time:
L(t)
Fluorescence Convolution:
F(t)= I(t)  L(t)
Example: HSA protein decay
 Nanosecond
flashlamp
excitation at
295nm
 Emission
detected at
340nm
 Three lifetimes
detected: 0.8ns,
3.6ns and
7.2ns.
HOT ns FLASHes!
JY-IBH Ltd. Announces the Launch of:
280 nm NanoLED
Facilitates ps work with tryptophan!
Huge savings over Ar and TiS lasers!
340 nm NanoLED
Replaces expensive Nitrogen lasers!
NanoLED
Pulsed laser diode and LED excitation sources
• (dashed) Laser Diodes emit
~100ps pulses
• (solid) LEDs emit ns pulses
NanoLED Sources
Pulse Widths
 Laser Diodes
 ~ 50ps – 150ps optical pulse FWHM
 Diode dependant: Typically red (635nm/650nm) diodes are faster than
violet, UV, blue, cyan
 N-07N high intensity 405nm source ~ 750ps
 LEDs
 New 280nm & 340nm 1ns
 All other LEDs ~ 1.0 – 1.4ns diode dependant
NanoLED Sources
Pulse Energies
Laser Diodes
Pulse energy
NanoLED-02
12 pJ nominal
NanoLED-02B
12 pJ nominal
NanoLED-2C
To be measured
NanoLED-07
20 pJ nominal
NanoLED-10
8pJ nominal
NanoLED-11
15 pJ nominal
NanoLED-12A/B
To be measured
NanoLED-14
8pJ nominal
LEDs
Pulse energy
NanoLED-01
1 pJ nominal
NanoLED-03
1 pJ nominal
NanoLED-04
2 pJ nominal
NanoLED-05
4 pJ nominal
NanoLED-06
2 pJ nominal
NanoLED-08
2 pJ nominal
NanoLED-09
0.07 pJ nominal
NanoLED-15
To be measured
NanoLED-16
To be measured
TBX Features
 Compact and integrated picosecond photon detection module
 Fast rise-time PMT with integral GHz timing preamplifier, constant fraction discriminator
and regulated HV supply
 Factory optimised
 Timing performance typically ~ 180ps (< 250ps guaranteed)
 Much cheaper and more robust than an MCP
 Photocathode sensitivity comparable to MCP
 9.5mm active area => easier to use than SPADs
 Easy to use “plug-and-play” operation:15V + Photons in  Logic pulses out
 NIM & TTL output signal
 Timing performance good enough for most applications (MCP upgrade available)
 Gold plated housing for maximum noise immunity
TBX Integrated Module
Power requirements 15V: TBX modules can be powered either from the
back of the DataStation HUB (un-cooled TBX-04 model only) or by the TBX-PS
TBX Models
All TBX models can be used on any JY-IBH system or sold as a component to
upgrade systems from other manufacturers
 TBX-04
 Spectral response 185nm-650nm
 Dark counts < 20cps typical, 80cps maximum
 TBX-05




Spectral response 300nm-850nm
Thermoelectrically cooled photocathode
Dark counts < 20cps typical
Recommend TBX-PS to power cooler
 TBX-06




Spectral response 185nm-850nm
Thermoelectrically cooled photocathode
Dark counts < 20cps typical
Recommend TBX-PS to power cooler
TBX Spectral Responses
Advantages of TCSPC
 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)