Fluorescence and Chemiluminescence - PowerPoint PPT Presentation

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Fluorescence and Chemiluminescence

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Title: Fluorescence and Chemiluminescence


1
Fluorescence andChemiluminescence
  • Skoumalová, Vytášek, Srbová

2
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
3
Fluorescence and fosforescence
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
5
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
6
Quantitative fluorescent measurement
Fluorescence efficiency (?f ) is the fraction of
the incident radiation which is emitted as
fluorescence ?f lt 1
7
Fluorescence measurement
Filter fluorimeters Spectrofluorometers
Fluorescent microscopes Fluorescent scanners
Flow cytometry
8
Spectrofluorometer
9
Spectrofluorometer
10
Analysis of the unknown sample
Erythrocytes (patients with Alzheimers disease)
11
Fluorescence microscopy
Endothelial cell (mitochondria, cytoskeleton,
nucleus)
12
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

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

14
Natural fluorophores
  • Polyaromatic hydrocarbons
  • Vitamin A, E
  • Coenzymes (FAD, FMN, NADH)
  • Carotenes
  • Quinine
  • Steroids
  • Aromatic aminoacids
  • Nucleotides
  • Fluorescent proteins GFP (green fluorescent
    protein)

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

16
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

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

18
Chemiluminiscence
19
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
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

peroxidase
21
Summary 1. The principle of fluorescence 2.
Applications of fluorescence in medicine -
examples 3. Chemiluminescence - applications
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