Fluorescence and Chemiluminescence

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Fluorescence andChemiluminescence

• Skoumalová, Vytášek, Srbová

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Luminescence

• 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
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Fluorescence and fosforescence
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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
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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/?

http//psych.lf1.cuni.cz/fluorescence/soubory/prin
cipy.htm
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Quantitative fluorescent measurement
Fluorescence efficiency (?f ) is the fraction of
the incident radiation which is emitted as
fluorescence ?f lt 1
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Fluorescence measurement
Filter fluorimeters Spectrofluorometers
Fluorescent microscopes Fluorescent scanners
Flow cytometry
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Spectrofluorometer
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Spectrofluorometer
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Analysis of the unknown sample
Erythrocytes (patients with Alzheimers disease)
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Fluorescence microscopy
Endothelial cell (mitochondria, cytoskeleton,
nucleus)
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Sources of interference

• Inner filter effect
• intensity of excitation light isnt 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
• Quenching
• 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

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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

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Natural fluorophores

• Polyaromatic hydrocarbons
• Vitamin A, E
• Coenzymes (FAD, FMN, NADH)
• Carotenes
• Quinine
• Steroids
• Aromatic aminoacids
• Nucleotides
• Fluorescent proteins GFP (green fluorescent
  protein)

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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

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Fluorescent probes

• Compounds whose fluorescence doesnt change after
  their interaction with biological material
• acridine orange (DNA)
• fluorescein (proteins)
• rhodamine (proteins)
• GFP
• Compounds whose fluorescence change according to
  their environment
• ANS (1-anilinonaftalen-8- sulphonate) -
  polarity
• Fura-2 - tracking the movement of calcium within
  cells

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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

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Chemiluminiscence
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Chemiluminescence

• Excitation of electrons is caused by chemical
  reaction
• Return to ground state is accompanied by light
  emission
• Bioluminescence

luciferase
ATP luciferin O2
AMP PPi CO2 H2O oxyluciferin light
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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

peroxidase
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Summary 1. The principle of fluorescence 2.
Applications of fluorescence in medicine -
examples 3. Chemiluminescence - applications