Fluorescence spectroscopy The light: electromagnetic wave Tamás Huber Biophysics seminar Dept. of Biophysics, University of Pécs 05-06. February 2014. 1
Luminescence: light emission of an excited system. From molecules or ions: molecular luminescence Basic phenomena are discribed by the Jablonski termscheme. 2
The types of luminescence Chemiluminescence - Photoluminescence 1. Chemiluminescence light emission that is excited by the energy from chemical reactions (e.g.: phosphor (P) oxidation) acceptable for examination of metabolisms low intensity depends on physiological relations 3
Bioluminescence: is the production and emission of light by a living organism as the result of a chemical reaction. Examples: firefly (bug), deep-sea fishes, medusa, octopus, bacteria, planktons 2. Photoluminescence Light emission that is exited by direct light radiation of certain energy (frequency) and wavelength. Very useful in molecular system assays, because it carries large amount of information of the properties of molecules, interactions and the relationship with its environment. 1. Luciferase catalizes the oxidation of luciferin. 2. Inactive oxyluciferin and light (h ) arise. 3. More luciferin replenish from food, or inner synthesis. Structure of luminescent molecules have aromatic rings with conjugated double bounds. Two types: fluorescence, phosphorescence 4
Jablonski termscheme The proof of the Kasha-rule: Any kind of excitation wavelength excites the molecule, the emission spectra does not change. Fluorescence: the molecule relaxes from the excited singlet state to the singlet ground state Lifetime: 10-9 s Phosphorescence: molecule relaxes from the excited triplet state to the singlet ground state (lower possibility) Lifetime: 10-6 -10 s http://www.olympusmicro.com/primer/java/jablonski/jabintro/ Separate them by: - the shape of the spectra, - the time interval of the excited state. 5
Characterization of the luminating material By its absorption spectrum and its fluorescence, phosphorescence excitation and emission spectrum Quantum yield of the radiation Lifetime of the excited state Polarization degree of the emission (anisotropy) Principles of measuring fluorescence The most important problem is to separate the excitation light and the caused luminescence light. Practical choice of the exciting and detecting directions Three different compositions 6
Sample 1. Detection is perpendicular to the direction of excitation. How to measure fluorescence? ( steady-state case) Sample 2. Detection is parallel to the excitation direction. Detection of the outcoming fluorescence from the front side. Sample 3. Detection from the opposite side to the excitation direction.!! Optical filters, monochromators!! 7
Intensity Intensity The excitation spectrum Detection at a fixed emission wavelength. Measuring the intensity as function of the excitation wavelength. The shape of its function is the same as the absorption spectrum. The emission spectrum Fluorescent emission spectrum Originates in the transition from the lowest vibrational level of the first singlet excitation state to one vibrational level of the ground state. Gives information of the vibrational levels of the ground state. Excitation Emission Excitation Emission Stokes-shift, mirror image spectra Wavelength Sir George Gabriel Stokes, 1 st Baronet (1819 1903) Wavelength 8
Fluorescence intensity, (a.u.) Effect of chemical denaturation to excitation and emission spectra Phosphorescence emission spectrum Excitation spectrum Phosphofescence emission spectrum Fluorescence emission spectrum GuHCl Wavelength, nm During the transformation from the first triplet excitation state to the singlet state. At room temperature only on crystal materials. According to the fluorescence spectrum its shifted towards to the infrared wavelengths. 9
Fluorescence lifetime Refers to the average time the molecule stays in its excited state before emitting a photon, or the number of excited photons decreases to the fraction e. = 1 / (k f + k sum ) f : fluorescence sum : f + vibr. + rot. (so, f + non-radiative) Quantum yield is the ratio of the number of photons emitted to the number of photons absorbed: Q = N em / N abs < 1 f fluorescence sum f + vibr. + rot. (so, f + non-radiative) - also expressible with the rate constants: Q = k f / (k f + k sum ) 10
Fluorescence Intensity (cps) How to measure lifetime? Time domain measurement Time-Correlated Single Photon Counting /TCSPC/ 1000 PEVK11 IAEDANS 100 10 short excitation pulses (~ fs) detection of photons in time windows Principles of Fluorescence Spectroscopy_Joseph R. Lakowicz. 1 1000 100 10 PEVK21 IAEDANS Principles of Fluorescence Spectroscopy_Joseph R. Lakowicz. 1 0 20 40 60 80 100 Time Domain Time (ns) 11
How to measure lifetime? Frequency domain measurement How to measure lifetime? Frequency domain measurement 12
Fluorescent dyes nativ or intrinsic fluorophores: Tryptophan, tyrosine, phenylalanine Advantage: no protein modification Extrinsic fluorophores Direct labeling with dyes: Dansyl Rhodamine IAEDANS IAF FITC Fluorescently labeled toxins: Falloidin B-scorpiontoxin A-bungarotoxin Macrophages Actin is labeled by phalloidin-alexa 568-cal (Red) Nuclei: DAPI (Blue) Streptococcus aureus (Green) 13
Labeling proteins with fluorescent dyes - quality and location can be planned - labeling is specific for the binding residues - protein could be modified, we have to test the activity 14
Labeling with specific antibodies (immunfluorescent, immunhistochemical labeling) The antibody binds to the surface of the recognized molecule with high affinity. Monoclonal and polyclonal antibodies. Primary antibody Antigen Fluorophore Secondary antibody Measuring phosphorescence The excitation light must be separated from the phosphorescence light in time The change of intensity during the time must be measurable. Must be measured at low temperature Phosphoroskop: After the excitation we hide the sample with an optical cylinder, then the emitted light can get to the detector. Direct labeling: a fluorescent dye is bound to the antibody Indirect labeling: the primary antibody is not labeled, the secondary antibody is labeled. Primary antibody Antigen The time after the excitation and before the detection depends on: the velocity of the rotation the number of the slits Shortest reachable time has a magnitude of 10-5 s. 15
Sample Sample The phosphoroscop The excitation light can get through the slit, but the phosphorescence can not get through the wall of the cylinder. After a quarter rotation the way of the excitation light is closed and the phosphorescence gets to the detector. The sample Usually a solution (protein, nucleic acid, pigment extract, cell suspension) The material of the cuvette must be nonfluorescent Glass cuvettes (visible range only) Special glass cuvettes (λ > 300 nm) Plastic cuvettes Special quartz cuvettes (measuring fluorescence) Cuvette holders: Temperature can be set More places (usually 4), rotatable 16
Excitation light sources Continuous-, (heated to high temperatures) Lamps filled with halogen gases Lamps filled with high pressure gases Optical filters Selection of different wavelengths Absorption filters Usually made of glass. Contains organic and inorganic components that is why light beams with given wavelength can go through and other wavelengths can not. Plastic (cheaper, lighter) Line-, (atoms) Intensive, monochromatic light Lamps filled with low pressure mercury Etc. Dicroic mirrors 17
Optical filters UV filters: UV rays can not go through, but rays with longer wavelength can. Neutral filters: Transmission has a wide spectrum range and independent from the wavelength. Photochemical, and photobiological processes can be examined. Interference filters If a thin transparent spacer is placed between two semireflective coatings, multiple reflections and interference can be used to select a narrow frequency band, producing an interference filter. Optical filters Long pass filters Allow to pass light with longer wavelengths. Fluorescence microscopy: dicroic mirrors usually used as emission filters. Short pass filters Optical interference or coloured glass filters. Allow to pass light with shorter wavelengths. Dicroic mirrors usually used as excitation filters. Band pass filters Combination of the uppers. Lower transmittance. Blocks everything beyond the chosen wavelengths. 18
Optical filters Monochromators A: Light source B: Slit C: Collimator D: Prism or grid E: Mirror F: Excitation slit G: Sample 19
The detector Photomultiplier tube: Very sensitive from the UV to the NIR. The End! Advantages of the application of fluorescence - Very good detection: measurable at low concentrations - Fluorescence is sensitive to the environment 20