Fluorescence and Chemiluminescence

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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
Fluorescence and Chemiluminescence

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Fluorescence and chemiluminescenceSlide 1

Fluorescence andChemiluminescence

Skoumalová, Vytášek, Srbová

LuminescenceSlide 2


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

Fluorescence and chemiluminescenceSlide 3

Fluorescence and fosforescence

Energy level diagram for photoluminescent moleculesSlide 4


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 shiftSlide 5

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 measurementSlide 6

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 measurementSlide 7

excitation monochromator



emission monochromator



Fluorescence measurement

Filter fluorimeters


Fluorescent microscopes

Fluorescent scanners

Flow cytometry

SpectrofluorometerSlide 8


Spectrofluorometer1Slide 9


Analysis of the unknown sampleSlide 10

Analysis of the unknown sample

Erythrocytes (patients with Alzheimer´s disease)

Fluorescence microscopySlide 11

Fluorescence microscopy

Endothelial cell (mitochondria, cytoskeleton, nucleus)

Sources of interferenceSlide 12

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 determinationSlide 13

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 fluorophoresSlide 14

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 2008Slide 15

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 probesSlide 16

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 detectionSlide 17

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

ChemiluminiscenceSlide 18

Luminol and peroxidase before adding H2O2

Chemiluminiscence after addition H2O2


ChemiluminescenceSlide 19


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 detectionSlide 20

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


Fluorescence and chemiluminescenceSlide 21


1. The principle of fluorescence

2. Applications of fluorescence in medicine - examples

3. Chemiluminescence - applications

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