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

Blue Excitation Absorbance energy

Internal Conversion Absorption femtoseconds 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

Whats 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 0.8 0.6 0.4 0.2 0 0 500 time, ps 1000

I(t)

=1/e=37%

Population of Molecules Excited With Instantaneous Flash

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
12000 12000

PHOTON COUNTS

10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 0
0

PHOTON COUNTS

Cumulative histogram

TIME, CHANNELS TIME, CHANNELS

statistical single photon events

nanoled S

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 15 19 14 18 13 17 12 16 11 15 10 14 9 13 8 12 7 11 6 10 5 9 4 8 3 7 2 6 1 5 4 3 2 1

TAC rate 1MHz Coaxial Delay 50 Ns Sync delay 20 ns

IBH 5000U

36 35 34 33

periodic pulses
SYNC CFD

2%
MCA

TAC

TCSPC Instrument Principle

100000 10000 PHOTON PHOTONCOUNTS COUNTS 1000 100 10 1 -10 -10 100000 100000 10000 PHOTON PHOTON COUNTS COUNTS 1000 100 10 1
18 19 20 0 1 6 7 7 8 8 9 9 10 10 11 11 12 13 14 15 16 17 18 19 20 1 2 2 3 3 4 4 5 5 6 1112 1213 1314 1415 1516 1617 17 18 19 20 TIME, CHANNELS TIME, CHANNELS CHANNELS

pulse decay

Time Domain Convolution Principle

Intensity as function of time: I(t)= exp (-t/)

-5 -5

0 0 TIME, CHANNELS TIME, CHANNELS

5 5

10 10

1 2 3 4

convolved decay

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 NanoLED-02 NanoLED-02B NanoLED-2C NanoLED-07 NanoLED-10 NanoLED-11 NanoLED-12A/B NanoLED-14 Pulse energy 12 pJ nominal 12 pJ nominal To be measured 20 pJ nominal 8pJ nominal 15 pJ nominal To be measured 8pJ nominal LEDs NanoLED-01 NanoLED-03 NanoLED-04 NanoLED-05 NanoLED-06 NanoLED-08 NanoLED-09 NanoLED-15 NanoLED-16 Pulse energy 1 pJ nominal 1 pJ nominal 2 pJ nominal 4 pJ nominal 2 pJ nominal 2 pJ nominal 0.07 pJ nominal To be measured 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)