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HORIBA Jobin Yvon Fluorescence Division Presents: Time-Resolved Fluorescence Spectroscopy Edison, NJ March 15, 2005 PowerPoint Presentation
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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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slide1
HORIBA Jobin Yvon Fluorescence Division Presents:Time-Resolved Fluorescence SpectroscopyEdison, NJMarch 15, 2005
fluorescence a type of light emission
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
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
slide5

Blue Excitation

Absorption

femtoseconds

S2 excited state

Internal Conversion

Absorbance energy

S1 excited state

Fluorescence

nanoseconds

Nonradiative

dissipation

Ground State

Electrons

basic principles of fluorescence
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
time resolved fluorescence
Time Resolved Fluorescence
  • What’s happening during the time of the fluorescence emission
  • Fluorescence Lifetime
what is a fluorescence lifetime

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

why measure lifetimes
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

Time Domain

TCSPC

Time Correlated Single Photon Counting

slide13

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

slide14

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

slide15

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
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
NanoLED Pulsed laser diode and LED excitation sources
  • (dashed) Laser Diodes emit ~100ps pulses
  • (solid) LEDs emit ns pulses
nanoled sources pulse widths
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
tbx features
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
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
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
advantages of tcspc
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)