Luminescence (Miklós Nyitrai; 27 th of February, 2007)

Energy levels

Transitions between levels

The Kasha rule

Time-scale of the changes

Definition of fluorescence and phosphorescence S T S S In the ns range In the > ms range

What is fluorescence spectra? Definition: the wavelength dependence of fluorescence emission a.Emission spectra b.Excitation spectra

Stokes shift, mirror image

Phosphorescence

Fluorescence quantum yield Rate constants!

Fluorescence lifetime

Radiation lifetime How to define fluorescence lifetime? Fluoresc. lifetime

How to measure fluorescence: the steady-state case

The advantages -great sensitivity and low detection limit -fluorophores are sensitive to the environment

How to measure fluorescence lifetime? Time Correlated Single Photon Counting Frequency Domain Principle Two basic common methods:

Time Correlated Single Photon Counting

After collecting the data make a fit!

A practical experiment (TCSPC)

Frequency Domain Principle

Comparison of the two methods nitrobenzoxadiazol

Light sources: lamps and lasers

Optical filters

Monochromators, polariser, Cuvettes, detectors

Classification of types of luminescence

The scheme of the reaction

Expected to be linear, but…! The inner-filter effect: Reabsorption of emitted fluorescence radiation Concentration dependence of fluorescence

Fluorescence probes, dyes: intrinsic fluorophores (definition) Tryptophan, tyrosin, phenilalanine Advantage: no modification of the protein is required.

Fluorescence probes, dyes: extrinsic fluorophores - e.g., dansyl, fluorescein, rhodamine, coumarin, lanthanides

Protein labelling -One can design labelled systems: flexibility and versality. -The fluorophores can be attached to specific sites. -The protein is modified, which can alter its properties.

Summary -The luminescence phenomena -The definition of fluorescence and phosphorescence -Fluorescence parameters -How to measure fluorescence? -Applications (see also next week)

The linearity of the spectrophotometer; “stray light effect” Observation:

The principles of a monochromator

The linearity of the spectrophotometer; “stray light effect” The origin of the problem: not perfect monochromators! Optical grating And the second- third… harmonics!!!! 2 ; 3 …

substance I0I0 I 99% chosen and 1% second h. Absorbs only at the chosen ! 89% chosen and 1% second h. In the case of low absorption.

substance I0I0 I 99% chosen and 1% second h. Absorbs only at the chosen ! 1% chosen and 1% second h. In the case of high absorption.

Low absorption: I / I 0 = 90 / 100 = 0.9 real absorption = 89 / 99 ~ 0.9 The measured is close to the real one! High absorption: I / I 0 = 2 / 100 = 0.02 real absorption = 1 / 99 ~ 0.01 Relatively large deviation! The transmitted light: measured vs. real at the chosen

The resulting effect