Luminescence spectroscopy

Transcription

1 Febr. 203 Luminescence spectroscopy Biophysics 2 nd semester Józse Orbán University o Pécs, Department o Biophysics Deinitions, laws FUNDAMENTALS o SPECTROSCY review - Spectral types (absorbtion/emission and line/band/continuous) - Absorbance (optical denzity, extinction), transmittance, extinction coeicient - Lambert-Beer law, additivity - Energy levels o atoms, photon energy resonance condition, LUMINESCENCE - Luminescence types (according to excitation and emission pathways) - Fluorescence and phosphorescence, singlet/triplet state, energetics - Time scale, coherence, polarisation, photoselection - Divergence, - Explanation o band type spectrum o molecules, molecular energetics Jablonsy term scheme, transitions - Mirror symmetry, Stoes shit - Lietime, quantum eiciency Deinitions, laws FLUORIMETER - DETECTION - Setup o a luorimeter: monochromators (excitation, emission), perpendicular arrangement, PMT Applications - Intrinsic / extrinsic luorophores - Molecular study; rom dynamic or static viewpoint - Investigations on molecular interactions Further applications topics o the ollowing lectures: - Anisotropy decay, FRET, luorescence decay, - luorescent microscopy Luminescence Some materials can emit photon spontaneously ater electron excitation by speciic method. Properties: Singlet/triplet state Energetics Time Orientation, coherence Polarisation Classiication o luminescence According to the excitation: EM radiation (bio)chemical reaction thermally activated induced by electric charge high energy particle or radiation mechanic (riction) sound waves According to the transition type: photoluminescence chemi-, bioluminescence thermoluminescence electroluminescence radioluminescence triboluminescence sonoluminescence singlet singlet transition luorescence triplet singlet transition phosphorescence Thermoluminescence is neither heat (IR) nor blac body radiation! 0 Ene ergy Total spin? s = S =? Singlet and triplet state Multiplicity: = 2 S+ S = 0 M = S = M = 3 S According to Pauli s principle permitted transition T Contradicts to Pauli s principle restricted transition

2 0 Energy Jabłonsy-type term scheme molecular system absorption time: 0-5 s S 2 S Emission. Excitation e - undergoes a transition between levels due to energy uptae. Photon is absorbed. 2. De-excitation excitation Emission Energy is released and the e - alls to a lower energy level. Photon is emitted. Kasha s rule (luorescence) All transition starts rom the lowest vibrational level o the irst excited singlet (S ) state. 0 En nergy Jabłonsy-type term scheme S 2 S IC: internal conversion VR: vibrational relaxation (thermal relaxation) ISC: intersystem crossing with spin turn Emission nonradiativ transitions T luorescence ( s) phosphorescence (0-6 0 s) SPONTANEOUS EMISSION! radiativ transitions Excitation Fluorescence emission spectra: mirror symmetry, Stoes shit ( ) How do we measure luorescence? Scheme o a luorimeter Int. : s p : s light source excitational monochromator Sample perpendicular excitationemission arrangement! abs. exc. luor. phosph. Light (excitation) Light (emission) Electric signal emission monochromator Detector Data aquisition and analysis (PC) Timescale o processes Fluorescence (average) lietime Fluorescence intensity decay 0-5 s relaxáció I ( t ) I 0 e t : rate constant (probability o transition) Fluorescence lietime is a characteristic parameter o luorophores, symbol: (tau) 2

3 Fluorescence (average) lietime ( ) An electron (molecule) returns to the ground state by emission o energy in the orm o photon (electromagnetic radiation). Fluorescence lietime is the time that the e-th portion o the excited electrons return to the ground state. ic where nr = ic + isc isc nr ns range Quantum eiciency o luorescence N Q N Q emit abs ic < isc ic isc - the probability or rate o the luorescence-transition ic - internal conversion isc - intersystem crossing nr - nonradiative N emit and N abs are not (or hardly) measurable, in contrast with and! Linearly (plane) polarised light Animation shows a vertically polarised electromagnetic wave. This animation shows a horizontally polarised wave. Linearly (plane polarised). View rom the perpendicular plane: I the electric ield s strength vector oscillates in only one plane all along the propagation then the wave is called linearly or plane polarised. I it is true in every point o a light beam, then the beam is polarised, as well. View rom the perpendicular plane Electric ield strength vector Electric dipole and absorbtion vectors Absorbtion vector Determines the probability o absorption. cone!!! absorption is maximal, i the absorption vector and the electric ield strength vector are parallel. absorption depends on cos 2 is the angle o the two vectors. Photoselection Fluorophores with randomly oriented absorption vector. unpolarised light vertically polarised light random, isotrop distribution Anisotrop distribution photoselection = selection o organised (directed) subpopulation o molecules by (polarised) light. 3

4 o proteins amino acids luorophores The three amino acids that absorb in UV, can be excited, so can emit as well. Extinction co oeicient wavelength Types o luorophores Chemical substance that can emit luorescence. Intrinsic, native luorophores Proteins: amino caids with aromatic side chain phe nyl ala nine tyrosine tryptophane Extrinsic luorophore: macromolecule luorophore chromophore luorophore The motion o the chromophore (luorophore) can be measured separately rom the macromolecule s motion. Covalently bound luorophores. Labeled antybodies. Other chemical derivates. macromolecule 2. Extrinsic luorophores - luorescent dyes The properties o the luorophore as a chemical agent, is easy to engineer (color, binding place). Speciic binding sites (in proteins) are: cystein, glutamin, lysin) Activity o the protein must be checed ater labeling! Intrinsic luorescence o cells IAF IAEDANS PYRENE Examples (or later studies): IAEDANS-IAF: FRET donor-acceptor pair Pyrene: to ollow actin polimerisation luorescein, dansyl, rhodamine derivatives... Actin monomer Aequorea victoria GFP (green luorescent protein) Modiications: YFP, BFP, CFP 4

5 excitation emission A series o luorophores Applications o Fluorescence Advantage Easy to detect (at low concentration o luorophores, as well). The properties o luorescence are sensible or (changes in) local environment. Useul tool o molecular level studies Perorms structural and dynamic inormation o the examined system (molecule or cell). Study o biological systems (cells, tissues): Intra- and intermolecular interactions. Molecular motion (by polarisation, anisotropy, FRET, FRAP). Distance o molecules (FRET). Flexibility o molecules (FRET). Exposure o protein domains (on surace or hidden, by quenching). Further applications: Supplement Structural properties can be studied Follow-up o protein denaturation Enzyme-ligand interaction Structural changes according to the changes in theenvironment t (Hi (ph, ions) Dynamic studies Reorganisations in the cell membrane or in organelles changes in the lexibility o protein cell matrix Properties o Emission type EM radiation and provided inormation on particles/interactions Stimulated With the incoming photon At the same time coherent Same direction (small divergence) Same wavelength monochromatic LASER Spontaneous (compared to the absorption) Delayed in time not coherent In all directions (3D) not identic band type Less energy LUMINESCENCE 5

6 Mirror symmetry Dependence o emission on applied excitation wavelength I we apply λ 2 (optimal excitation wavelength) then a large amount o photon will be absorbed and as a consequence, large amount o electron will be excited in the sample. This results in high intensity emission (in a wide spectral range). I I we apply λ (non optimal exc. wavelength) then less photon will be absorbed, less electron will be excited. Lower intensity emission is the response. BUT! The emission s spectral range do not change. explanation: probability, Kasha s rule Transitions happen in both case rom the same level, to various vibrational levels. λ λ 2 λ (nm) 6