1. HORIBA Jobin Yvon Fluorescence Division Presents: Time-Resolved Fluorescence Spectroscopy Edison, NJ March 15, 2005

2. Fluorescence: a type of light emission First observed from quinine by Sir J. F. W. Herschel in 1845First observed from quinine by Sir J. F. W. Herschel in 1845 Blue glass Filter Church Window! <400nm Quinine Solution Yellow glass of wine Em filter > 400 nm 1853 G.G. Stoke coined term “fluorescence”

3. 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

4. Common Fluorophores

5. Ground State Electrons S 1 excited state S 2 excited state Absorbance energy Fluorescence nanoseconds Absorption femtoseconds Nonradiative dissipation Blue Excitation Internal Conversion

6. 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

7. “Stokes” shift Absorption vs Emission  E = hc /

8. Time Resolved Fluorescence What’s happening during the time of the fluorescence emission Fluorescence Lifetime

9. 0 0.2 0.4 0.6 0.8 1 05001000 time, ps I(t) What is a Fluorescence Lifetime? Population of Molecules Excited With Instantaneous Flash Random Decay Back to Ground State: Each Molecule Emits 1 Photon  =1/e=37%

10. 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

11. Time Domain TCSPC Time Correlated Single Photon Counting

13. MCA S CFD SYNC IBH 5000U TAC rate 1MHz Coaxial Delay 50 Ns Sync delay 20 ns TBX-04 nanoled  ≤ 2% statistical single photon events periodic pulses Cumulative histogram TACV TCSPC Instrument Principle

14.            pulse decay Intensity as function of time: I(t)=  exp (-t/  ) Lamp intensity as function of time: L(t) Fluorescence Convolution: F(t)= I(t)  L(t) convolved decay Time Domain Convolution Principle

15. Example: HSA protein decay  Nanosecond flashlamp excitation at 295nm  Emission detected at 340nm  Three lifetimes detected: 0.8ns, 3.6ns and 7.2ns.

16. HOT ns FLASHes! 280 nm NanoLED Facilitates ps work with tryptophan! Huge savings over Ar and TiS lasers! 340 nm NanoLED Replaces expensive Nitrogen lasers! JY-IBH Ltd. Announces the Launch of:

17. NanoLED NanoLED Pulsed laser diode and LED excitation sources (dashed) Laser Diodes emit ~100ps pulses (solid) LEDs emit ns pulses

18. 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

19. NanoLED Sources Pulse Energies Laser DiodesPulse energy NanoLED-0212 pJ nominal NanoLED-02B12 pJ nominal NanoLED-2CTo be measured NanoLED-0720 pJ nominal NanoLED-108pJ nominal NanoLED-1115 pJ nominal NanoLED-12A/BTo be measured NanoLED-148pJ nominal LEDsPulse energy NanoLED-011 pJ nominal NanoLED-031 pJ nominal NanoLED-042 pJ nominal NanoLED-054 pJ nominal NanoLED-062 pJ nominal NanoLED-082 pJ nominal NanoLED-090.07 pJ nominal NanoLED-15To be measured NanoLED-16To be measured

20. 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

21. 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

22. TBX Models  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 All TBX models can be used on any JY-IBH system or sold as a component to upgrade systems from other manufacturers

23. TBX Spectral Responses

24. 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)