Fluorescence and Chemiluminescence Skoumalová, Vytášek, Srbová

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 S 0 S 1 T 1 E Singlet 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 S 1  S 0 probability of fluorescence is higher than phosphorescence Phosphorescence – transition between states with different multiplicity T 1  S 0

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

Quantitative fluorescent measurement I0I0 ItIt IfIf intensity of fluorescence I f intensity of absorptionI a  f = = I a = I 0 - I t sample Fluorescence efficiency (  f ) is the fraction of the incident radiation which is emitted as fluorescence  f < 1

Fluorescence measurement sample emission monochromator detector source excitation monochromator Read-out Filter fluorimeters Spectrofluorometers Fluorescent microscopes Fluorescent scanners Flow cytometry

Spectrofluorometer

Analysis of the unknown sample Erythrocytes (patients with Alzheimer´s disease)

Fluorescence microscopy Endothelial cell (mitochondria, cytoskeleton, nucleus)

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: O 2, 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 H 2 O 2 Chemiluminiscence after addition H 2 O 2

Chemiluminescence Excitation of electrons is caused by chemical reaction Return to ground state is accompanied by light emission Bioluminescence firefly Noctiluca scintillans ATP + luciferin + O 2 AMP + PPi + CO 2 + H 2 O + oxyluciferin + light luciferase

Application of chemiluminescence detection NO assay NO + O 3  NO 2 * + O 2 NO 2 *  NO 2 + light H 2 O 2 assay, peroxidase activity assay, immunochemical assays Luminol + H 2 O 2 3-aminoftalate + light peroxidase

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