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Fluorescence and Chemiluminescence Richard Vytášek 2008 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 Other type of luminiscence – e.g. triboluminiscence, catodoluminiscence, radioluminiscence Singlet state - spins of two electrons are paired E Triplet state - spins of two electrons are unpaired S0 S1 T1 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 Phosphorescence – transition between states with different multiplcity T1S0 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/ Stokes´ shift http://psych.lf1.cuni.cz/fluorescence/soubory/principy.htm Quantitative fluorescent measurement I0 sample If It f = Ia = I0 - It intensity of fluorescence If intensity of absorption Ia = Fluorescence efficiency (f ) is the fraction of the incident radiation which is emitted as fluorescence f < 1 If = f .Ia = f (I0 - It) If = f .I0(1- 10 It = I0 .10-ecd -ecd ) ecd < 0,01 10-ecd ln10.(1- ecd) If = 2,3.f.e.d.c = .c The dependence of intensity of fluorescence to concentration of fluorophore Fluorescence measurement Filter fluorimeters Spectrofluorometers Fluorescent microscopes Fluorescent scanners Flow cytometry source excitation monochromator sample emission monochromator detector Read-out 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 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 • • • • • • • • • • Lanthanides 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 emission Bioluminescence firefly Noctiluca scintillans ATP + luciferin + O2 luciferase AMP + PPi + CO2 + H2O + oxyluciferin + light Application of chemiluminescence detection • NO assay NO + O3 NO2* + O2 NO2* NO2 + light • H2O2 assay, peroxidase activity assay, immunochemical assays Luminol + H2O2 peroxidase 3-aminoftalate + light Thank you for your attention
