Fluorescence and Chemiluminescence Skoumalová, Vytášek, Srbová.

FluorescenceFluorescence and and

ChemiluminescenceChemiluminescence

Skoumalová, Vytášek, Srbová

LuminescenceEmission 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

S0 S1 T1

ESinglet state - spins of two electrons are paired

Triplet state - spins of two electrons are unpaired

Fluorescence and fosforescence

Energy level diagram for photoluminescent molecules

Radiationless transitions:

VR –vibrational relaxation

IC- internal conversion

ISC –intersystem crossing

E

Radiation transitions:

Fluorescence - transition to the ground state with the same multiplicity S1S0

probability of fluorescence is higher than phosphorescence

PhosphorescencePhosphorescence – – transition between states with different multiplicity Ttransition between states with different multiplicity T11SS00

Stokes shift

Wavelength of emitted radiation is longer because its energy is lower

E = h . c/

Stokes´ shift

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

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

Quantitative fluorescent measurement I0 It

If

intensity of fluorescence If

intensity of absorption Ia

f = =

Ia = I0 - It

sample

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

Fluorescence measurement

sample

emission monochromator

detector

sourceexcitation

monochromator

Read-out

Filter fluorimeters

Spectrofluorometers

Fluorescent microscopes

Fluorescent scanners

Flow cytometry

Spectrofluorometer

Spectrofluorometer

Analysis of the unknown sample

Erythrocytes (patients with Alzheimer´s disease)

Fluorescence microscopy

Endothelial cell (mitochondria, cytoskeleton, nucleus)

Sources of interferenceInner 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

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

• 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

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

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

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

Chemiluminiscence

Luminol and peroxidase before adding H2O2

Chemiluminiscence after addition H2O2

Chemiluminescence• Excitation of electrons is caused by chemical

reaction • Return to ground state is accompanied by light

emissionBioluminescence

firefly

Noctiluca scintillans

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

Application of chemiluminescence detection

• NO assay

NO + O3 NO2* + O2

NO2* NO2 + light

• H2O2 assay, peroxidase activity assay, immunochemical assays

Luminol + H2O2 3-aminoftalate + lightperoxidase

Summary:

1. The principle of fluorescence

2. Applications of fluorescence in medicine - examples

3. Chemiluminescence - applications