Luminescence (Miklós Nyitrai; 27 th of February, 2007)
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Transcript of Luminescence (Miklós Nyitrai; 27 th of February, 2007)
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Luminescence(Miklós Nyitrai; 27th of February, 2007)
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Energy levels
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Transitions between levels
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The Kasha rule
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Time-scale of the changes
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Definition of fluorescence and phosphorescence
S
T
S
S
In the ns range
In the > ms range
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What is fluorescence spectra?
Definition: the wavelength dependence of fluorescence emission
a. Emission spectra
b. Excitation spectra
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Stokes shift, mirror image
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Phosphorescence
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Fluorescence quantum yield
Rate constants!
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Fluorescence lifetime
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Radiation lifetime
How to define fluorescence lifetime?
Fluoresc. lifetime
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How to measure fluorescence: the steady-state case
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The advantages
-great sensitivity and low detection limit
-fluorophores are sensitive to the environment
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How to measure fluorescence lifetime?
Time Correlated Single Photon Counting
Frequency Domain Principle
Two basic common methods:
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Time Correlated Single Photon Counting
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After collecting the data make a fit!
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A practical experiment (TCSPC)
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Frequency Domain Principle
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Comparison of the two methods
nitrobenzoxadiazol
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Light sources: lamps and lasers
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Optical filters
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Optical filters
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Monochromators,
polariser,
Cuvettes,
detectors
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Classification of types of luminescence
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The scheme of the reaction
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Expected to be linear, but…!
The inner-filter effect: Reabsorption of emitted fluorescence radiation
Concentration dependence of fluorescence
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Fluorescence probes, dyes: intrinsic fluorophores
(definition)
Tryptophan, tyrosin, phenilalanine
Advantage: no modification of the protein is required.
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Fluorescence probes, dyes: extrinsic fluorophores
- e.g., dansyl, fluorescein, rhodamine, coumarin, lanthanides
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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.
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Summary
-The luminescence phenomena
-The definition of fluorescence and phosphorescence
-Fluorescence parameters
-How to measure fluorescence?
-Applications (see also next week)
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The linearity of the spectrophotometer;
“stray light effect”
Observation:
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The principles of a monochromator
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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…
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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.
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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.
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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
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The resulting effect