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Fluorescence and Chemiluminescence. Skoumalová, Vytášek, Srbová. E. S 0 S 1 T 1. Luminescence. Emission of radiation, which occurs during returning of excitated molecules to ground state

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Fluorescence and chemiluminescence

Fluorescence andChemiluminescence

Skoumalová, Vytášek, Srbová



S0 S1 T1


Emission of radiation, which occurs during returning of excitated molecules to ground state

Fluorescence, phosphorescence – excitation is caused by absorption of radiation

Chemiluminiscence – excitation is caused by chemical reaction

Singlet state - spins of two electrons are paired

Triplet state - spins of two electrons are unpaired

Energy level diagram for photoluminescent molecules


Energy level diagram for photoluminescent molecules

Radiationless transitions:

VR –vibrational relaxation

IC- internal conversion

ISC –intersystem crossing

Radiation transitions:

Fluorescence - transition to the ground state with the same multiplicity S1S0

probability of fluorescence is higher than phosphorescence

Phosphorescence – transition between states with different multiplicity T1S0

Stokes shift

Stokes´ shift

Stokes shift

Wavelength difference between absorption (excitation) and fluorescence (emission) maximum

Wavelength of emitted radiation is longer because its energy is lower

E = h . c/


Quantitative fluorescent measurement

intensity of fluorescence If

intensity of absorption Ia




f =


Ia = I0 - It


Quantitative fluorescent measurement

Fluorescence efficiency (f ) is the fraction of the incident radiation which is emitted as fluorescence f < 1

Fluorescence measurement

excitation monochromator



emission monochromator



Fluorescence measurement

Filter fluorimeters


Fluorescent microscopes

Fluorescent scanners

Flow cytometry

Analysis of the unknown sample
Analysis of the unknown sample

Erythrocytes (patients with Alzheimer´s disease)

Fluorescence microscopy
Fluorescence microscopy

Endothelial cell (mitochondria, cytoskeleton, nucleus)

Sources of interference
Sources of interference

Inner filter effect

intensity of excitation light isn´t constant because each layer of the sample absorbs some of the incident radiation (intensity of exciting light is higher in the front part of cuvette and lower in the rear part of cuvette


excited molecule returns to the ground state by radiationless transition (without emitting light) as a result of a collision with quenching molecule

Quenching agents: O2, halogens (Br, I), nitrocompounds

Methods of fluorescence determination
Methods of fluorescence determination

Direct methods - natural fluorescence of the fluorecent sample is measured

Indirect (derivatisation) methods - the nonfluorescent compound is converted into a fluorescent derivative by specific reaction or marked with fluorescent dye by attaching dye to the studied substance

Quenching methods - analytical signal is the reduction in the intensity of some fluorescent dye due to the quenching action of the measured sample

Natural fluorophores
Natural fluorophores

  • Polyaromatic hydrocarbons

  • Vitamin A, E

  • Coenzymes (FAD, FMN, NADH)

  • Carotenes

  • Quinine

  • Steroids

  • Aromatic aminoacids

  • Nucleotides

  • Fluorescent proteins –GFP (green fluorescent protein)

Nobel prize in chemistry in 2008
Nobel prize in chemistry in 2008

Osamu Shimomura discovered green fluorescent protein (GFP) in the small glowing jellyfish Aequorea victoria

Martin Chalfie introduced using of green fluorescent protein as a marker for gene expression

Roger Y. Tsien engineered different mutants of GFP with new optical properties (increased fluorescence, photostability and a shift of the major excitation peak ) and contributed to the explanation of mechanismus of GFP fluorescence

Fluorescent probes
Fluorescent probes

Compounds whose fluorescence doesn´t change after their interaction with biological material

acridine orange (DNA)

fluorescein (proteins)

rhodamine (proteins)


Compounds whose fluorescence change according to their environment

ANS (1-anilinonaftalen-8- sulphonate) - polarity

Fura-2 - tracking the movement of calcium within cells

Some applications of fluorescence detection
Some applications of fluorescence detection

  • Protein conformation

  • Membrane potential

  • Membrane transport

  • Membrane viscosity

  • Enzymatic reactions

  • DNA analysis

  • Genetic engineering (manipulations)

  • Immunochemical methods

  • Cell proliferation and apoptosis


Luminol and peroxidase before adding H2O2

Chemiluminiscence after addition H2O2




Noctiluca scintillans


  • Excitation of electrons is caused by chemical reaction

  • Return to ground state is accompanied by light emission



ATP + luciferin + O2 AMP + PPi + CO2 + H2O + oxyluciferin + light

Application of chemiluminescence detection
Application of chemiluminescence detection

  • NO assay

    NO + O3 NO2* + O2

    NO2*  NO2 + light

  • H2O2 assay, peroxidase activity assay, immunochemical assays

    Luminol + H2O2 3-aminoftalate + light



1. The principle of fluorescence

2. Applications of fluorescence in medicine - examples

3. Chemiluminescence - applications