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Basics of Fluorescence 2019

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Basics of Fluorescence 2019 Basics of fluorescence Microscopy Course IST Austria, 24.09.- 1.10. 2019 Anna Hapek-Sikora Emission of light- LUMINESCENCE Luminescence is the emission of light from any substance not resulting from heat and occurs from electronically excited states. According to excitation mechanisms: - chemiluminescence - thermoluminescence - electroluminescence and... PHOTOLUMINESCENCE PHOTOLUMINESCENCE fluorescence phosphorescence occures from excited singlet states occures from excited triplet states emission rates slow 103 – 100 s-1 emission rates very fast 109 s-1 lifetimes miliseconds to seconds lifetimes 10 ns Common fluorophores Common fluorophores • organic molecules with aromatic ring structures • delocalized electrones in binding π-orbitals • π-electrons interact with the environment What we see, what we cannot see E = hν = hc/λ Few people you should know Sir George Stokes Max Planck Nobel prize in 1918 Sir John Herschel Aleksander Jabłoński But we can try Good enough ;) Jabłoński Energy Diagram Stokes Shift Different deactivation pathways, through which an excited molecule can return to its ground-state. Characteristic of fluorescence emission ● the Stokes' shift ● emission spectra are independent of excitation λ ● fluorescence quantum yield ● fluorescence lifetime ● fluorescence quenching Stokes' shift Absorption and emission spectra of Rhodamine with ~25 nm Stokes shift Emission spectra are independent of excitation λ The emission spectrum is independent of the excitation energy as a consequence of rapid internal conversion from higher initial excited states to the lowest vibrational energy level of the S(1) excited state. Quantum yield Ratio of the number of emitted to absorbed photons S1 Relaxation (10 -12s) S1 Q= Γ • ᴦ - emissive rate • knr - non radiative decay rate to S0 hϑA hϑA ᴦ knr Γ +k nr S0 • Q is close to unity when knr ˂ ᴦ • substances with largest quantum yields (rhodamins) display brightest emissions • Q is highly dependent on the solvent of the fluorophore Substances with the largest quantum yield display the brightest emissions. Quantum yield of different fluorophores Fluorescence lifetime (τ) - τ is the average value of the time spent in the excited state - formally, the fluorescence lifetime is defined as the time in which the initial fluorescence intensity of a fluorophore decays to 1/e (approximately 37 percent) of the initial intensity - determines time avaiable for molecular interactions, diffusion into environment and hence the info avaiable from its emission F(t) = F0 • exp(-t/τ) 1 τ= ᴦ + knr • ᴦ - emissive rate • knr - non radiative decay rate to S0 τ Fluorescence quenching Refers to any process which decreases the fluorescence intensity of a given substance. Different mechanisms: Collisional quenching (dynamic) - excited state fluorophore is deactivated by other molecules (quenchers) - - - - O2; I , Cl (halogens); amines, e deficient molecules - mechanisms: e- transfer ( Indole → Acrylamide) - intersystem crossing to triplet state (halogens, heavy atoms) Static quenching - fluorophore forms nonfluorescent complexes with other molecules - occures in the ground state independent of diffusion or molecule collision (dye aggregation ) Nonmolecular mechanisms - attenuation of the incident light Fluorescence Anisotropy Effects of polarized excitation and rotational diffusion on the polarization or anisotropy of the emission. Photoselected Randomized fluorophores fluorophores Rotational diffusion I III polarized polarized unpolarized excitation emission emission (laser light) Resonance energy transfer - occurs in excited state - whenever emission spectrum of fluorophore called DONOR overlaps with absorption spectrum of another molecule called ACCEPTOR Förster resonance energy transfer FRET FRET occurs when DONOR and ACCEPTOR are within the Förster distance (0.5-10 nm) Donor-Acceptor spectral overlap FRET efficiency The FRET efficiency (E) is the quantum yield of the energy transfer transition, i.e. the fraction of energy transfer event occurring per donor excitation event. 1 EFRET – efficiency EFRET = 6 r – distance donor-acceptor R - the distance at which the energy transfer efficiency is 50% 1+(r /R0) 0 R0 50% of E FRET efficiency The efficiency of the energy transfer decreases to the 6th power of the distance between donor and acceptor. Donor Acceptor Donor Acceptor Förster Fluorescent Excitation Emission Quantum Extinction Distance Protein Maximum Maximum Yield Coefficient (nm) Pair (nm) (nm) EBFP2-mEGFP 383 507 0.56 57,500 4.8 ECFP-EYFP 440 527 0.40 83,400 4.9 Cerulean-Venus 440 528 0.62 92,200 5.4 MiCy-mKO 472 559 0.90 51,600 5.3 TFP1-mVenus 492 528 0.85 92,200 5.1 CyPet-YPet 477 530 0.51 104,000 5.1 EGFP-mCherry 507 510 0.60 72,000 5.1 Venus-mCherry 528 610 0.57 72,000 5.7 Venus-tdTomato 528 581 0.57 138,000 5.9 Venus-mPlum 528 649 0.57 41,000 5.2 FRET applications ● can be used to measure distances between domains in a single protein and therefore to provide information about protein conformation ● detect interaction between proteins ● applied in vivo, FRET has been used to detect the location and interactions of genes and cellular structures including intergrins and membrane proteins ● is also used to study lipid rafts in cell membranes Summary Molecular information from fluorescence Emission spectra and Stoke’s shift - location of the fluorophore - binding to macromolecules (DNA, membrane) - chemical sensing dyes Quenching of fluorescence - accessibility of fluorophores to quenchers (location on macromolecule, porosity of proteins) FRET - distance measurment between sites on macromolecules. Protein-protein interaction. Fluorescence polarization and anisotropy - used to measure molecular weight of proteins or protein binding 10 minutes break! Fluorophores Intrinsic Extrinsic natural added to the sample aromatic aa fluorescein NADH rhodamine, flavins DAPI chlorophyll Cells are autofluorescent! Important characteristics of fluorophores ● Absorbtion and emission maxima (nm) ● Fluorescensce intensity of the emitted light ● Molar Extinction Coefficient ● Quantum Yield (0-1) ● Solubility in water or other substances ● Lifetime of the excited state ● Photostability Absorbtion and emission maxima Molar extinction coefficients M-1 cm-1 The extinction coefficient is determined by measuring the absorbance at a reference wavelength (characteristic of the absorbing molecule) for a one molar (M) concentration (one mole per liter) of the target chemical in a cuvette having a one-centimeter path length. Extinction coefficients are a direct measure of the ability of a fluorophore to absorb light, and those chromophores having a high extinction coefficient also have a high probability of fluorescence emission. Molecules exhibiting a high extinction coefficient have an excited state with a short intrinsic lifetime. Photostability and photobleaching Fluorophores permanently loses ability to fluorescence. Why? The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent upon the molecular structure and the local environment. - due to photon induced chemical damage - covalent modifications - transition from singlet to triplet state (longer time in excited state to interact with others molecules) Dye + dye photobleaching Dye+light+oxygen How to control photobleaching • reduction of light intensity and time span of light exposure • increase of fluorophore concentration • remove oxygen • stop (compete with) photooxidation use antioxidants „antifade reagents“ (e.g. DABCO) • 2-photon excitation • employment of more robust fluorophores (Alexa Fluor dyes) • employment of dyes with longer excitation wavelength FRAP – application of photobleaching Used to investigate the diffusion and motion of biological macromolecules. Phototoxicity Cell damage as a result of exciting exogenous or endogenous fluorophores with lasers or high-intensity arc-discharge lamps Reasons: • In their excited state, fluorescent molecules tend to react with molecular oxygen to produce free radicals that can damage subcellular components • particular constituents of standard culture media, including the vitamin riboflavin and the amino acid tryptophan, may also contribute to adverse light-induced effects on cultured cells • interaction between light and DNA intercalating dyes causes denaturation of the polymer, Bernas T. et al. 2005 How to fight with it? Fighting with photoxicity Fighting with photoxicity • reduction of light intensity and time span of light exposure • addition of scavengers into the medium (ascorbic acid) • fluorescent proteins are generally not phototoxic • fluorophores that exhibit the longest excitation wavelengths possible should be chosen to minimize damage to cells by short wavelength illumination • many synthetic fluorophores, such as the MitoTracker and nucleic acid stains (Hoechst, SYTO cyanine dyes, and DRAQ5) are highly toxic to cells. Good to read And many more, just google it Antibody labeling Covalent binding: Noncovalent binding: • free NH2-groups • Fc-portion of antibodies • free SH-Groups ( Zenon® labeling system) • carbohydrate side chains Antibody labeling Protein labeling reagents Rhodamine FITC 600/615 480/510 Why this two? - high quantum yield (0.90, 0.95) - display longer excitation and emission λ than aa - high molar extinction coefficient (80 000 M-1 cm-1) - lifetime ~ 4ns - good for antibody labeling Stokes shift in protein labeling FITC 494/519 Small stoke shift leads to self-quenching events FRET can occur between two molecules attached to
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