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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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Slide 1

Fluorescence andChemiluminescence

Skoumalová, Vytášek, Srbová

Slide 2

E

S0 S1 T1

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

Slide 3

Fluorescence and fosforescence

Slide 4

E

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

Slide 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/

http://psych.lf1.cuni.cz/fluorescence/soubory/principy.htm

Slide 6

intensity of fluorescence If

intensity of absorption Ia

I0

It

sample

f =

=

Ia = I0 - It

If

Quantitative fluorescent measurement

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

Slide 7

excitation monochromator

sample

source

emission monochromator

detector

Read-out

Fluorescence measurement

Filter fluorimeters

Spectrofluorometers

Fluorescent microscopes

Fluorescent scanners

Flow cytometry

Slide 8

Spectrofluorometer

Slide 9

Spectrofluorometer

Slide 10

Analysis of the unknown sample

Erythrocytes (patients with Alzheimer´s disease)

Slide 11

Fluorescence microscopy

Endothelial cell (mitochondria, cytoskeleton, nucleus)

Slide 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

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

Slide 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

Slide 14

Natural fluorophores

  • Polyaromatic hydrocarbons

  • Vitamin A, E

  • Coenzymes (FAD, FMN, NADH)

  • Carotenes

  • Quinine

  • Steroids

  • Aromatic aminoacids

  • Nucleotides

  • Fluorescent proteins –GFP (green fluorescent protein)

Slide 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

Slide 16

Fluorescent probes

Compounds whose fluorescence doesn´t 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

Slide 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

Slide 18

Luminol and peroxidase before adding H2O2

Chemiluminiscence after addition H2O2

Chemiluminiscence

Slide 19

firefly

Noctiluca scintillans

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

Slide 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

Slide 21

Summary:

1. The principle of fluorescence

2. Applications of fluorescence in medicine - examples

3. Chemiluminescence - applications


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