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Fluorescence: a type of light emission

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.

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Fluorescence: a type of light emission

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  1. HORIBA Jobin Yvon Fluorescence Division Presents:Time-Resolved Fluorescence SpectroscopyEdison, NJMarch 15, 2005

  2. 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

  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. Blue Excitation Absorption femtoseconds S2 excited state Internal Conversion Absorbance energy S1 excited state Fluorescence nanoseconds Nonradiative dissipation Ground State Electrons

  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” shiftAbsorption vs Emission E = hc /   

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

  9. 1 0.8 0.6 t=1/e=37% I(t) 0.4 0.2 0 0 500 1000 time, ps What is a Fluorescence Lifetime? Random Decay Back to Ground State: Each Molecule Emits 1 Photon Population of Molecules Excited With Instantaneous Flash

  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

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

  13. Time Domain Convolution Principle d-pulse decay Intensity as function of time: I(t)=a exp (-t/t) d 0 convolved decay d-1 Lamp intensity as function of time: L(t) d-2 d-3 Fluorescence Convolution: F(t)= I(t)  L(t) d-4

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

  15. 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!

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

  17. NanoLED SourcesPulse 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

  18. NanoLED Sources Pulse Energies

  19. 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

  20. 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

  21. 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

  22. TBX Spectral Responses

  23. 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)

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