Post on 18-Dec-2015
Luminescence(Miklós Nyitrai; 27th 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
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
I0 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
I0 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 / I0 = 90 / 100 = 0.9 real absorption = 89 / 99 ~ 0.9
The measured is close to the real one!
High absorption:
I / I0 = 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