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Exploring Fluorescent Dye Probes: Applications and Techniques

Discover the world of fluorescent dye probes in optical spectroscopy, their functions, and applications in providing information on local environments. Learn about chemosensors, time-correlated single photon counting, and fluorescence quantum yield determination. Dive into fluorescence imaging, molecular structures, and spectroscopic methods for chiral compounds.

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Exploring Fluorescent Dye Probes: Applications and Techniques

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  1. OpticalSpectroscopy 2018 • Fluorescentdyeprobes(KM), 26 Sept • Photochromicmaterials(BP), 3 Oct • Luminescent metal complexes(BP), 10 Oct • Photodynamictherapy (VT), 17 Oct • Fluorescenceimaging(VT), 24 Oct • NearInfraredSpectroscopy (GSz) 31 Oct • Spectroscopy of chiralcompounds(KM), 7 Nov

  2. ALAPISMERETEK(vizsgára, doktori szigorlatra átismételni) Kémiai anyagszerkezettan V. OPTIKAI SPEKTROSZKÓPIA (Optsp05) VI. A MOLEKULÁK FORGÓMOZGÁSA (Forgo05) VII. A MOLEKULÁK REZGŐMOZGÁSA (Rezgo05) VIII. A MOLEKULÁK ELEKTRONSZERKEZETE (Molel05) X. LÉZEREK, LÉZERSPEKTROSZKÓPIAI MÓDSZEREK (Lezer05)

  3. Nobel prize in Chemistry 2008 GFP = Green Fluorescent Protein Martin Chalfie Osamu Simamura Roger Y. Tsien

  4. Nobel prize in Chemistry 2014 STED = Stimulated Emission Depletion Eric Betzig William E. Moerner Stefan W. Hell

  5. Jablonski-diagram

  6. Advantages of detection of fluorescence over detection of absorbtion 1. Sample may be non-transparent 2. Higher sensitivity 3. Triple selectivity - excitation wavelength - emission wavelength - delay time Disadvantage: only a few type compounds are fluorescent

  7. Fluoreszcent dye probesFunction: provide information on local environment J. R. LAKOWICZ, Principles of Fluorescence Spectroscopy, 2nd Edition, Kluwer Academic, London, 1999

  8. Main points •  Instruments • stationaryfluorescencespectrometer • time-correlatedsinglephotoncounting • Molecularchemosensors: detection of ions, molecules •  Polaritysensors • Viscositysensors •  Fluorescence of proteins / triptophan • Distancemeasurement : FRET

  9. Spectrofluorimeters • stationary • time-resolved (measures F, time-correlated single photon counting)

  10. Stacionárius

  11. Excitation and emission flurescence spectra Excitation sp: similar to absorption sp, bands of S0 →S1, S0 →S2, ∙∙∙ transitions Emission sp: only S1 →S0, IF is relative, depends on instrument! a.u.!

  12. Fluorescence quantum yield Determination of F - integrating sphere - standard X: sample R: standard IX,IR integrated intensities of fluorescence bands AX, ARabsorbances at excitation wavelength nX, nRindeces of refraction

  13. Time-correlated single photon counting

  14. Fluorescence decay curve IRF

  15. Chemosensors: fluorescent detection of non-fluorescent ions, molecules

  16. Chemosensor for fluorescent detection of Na+ ions CoroNa Green

  17. Chemosensor for fluorescent detection of Na+ ions Distribution of Na+ ions eloszlása neurons, Microscopic image with application of CoroNa Green W. J. Tyler et al. , PlosOne 3, e3511 (2008)

  18. Chemosensor for fluorescent detection of Cl- ions MQAE Selective: nitrate, phosphate – no quenching, Br-, I- quenching Its operation is based on dynamic quenching

  19. Chemosensor for fluorescent detection of Cl- ions Distribution of Cl- ions in neurons IF image FLIM: fluorescence lifetime imaging

  20. Dynamic quenching: Stern-Volmer equation M + h M + h M +  M* M + Q

  21. Deactivation rates and fluorescence quantum yields in the absence and presence of quencher No quencher With quencher

  22. Stern-Volmer equation

  23. Polarity sensors Nile red water, - methanol - ethanol - acetonitrile -dimethylformamide, 6. acetone -ethyl acetate - dichloromethane - n-hexane - methyl-tert-butylether - cyclohexane - toluene.

  24. Solvatochromic dye: color depends on solvent

  25. Confocal image of the algae stained by Nile red The accumulation of oil droplets (golden dots). Red represents chlorophyll autofluorescence. phys.org

  26. „charge transfer (CT)” dyes S1 S0 solvent polarity

  27. - - - - + G v. E _ + + + + 2a Effect of polarity on spectra: Lippert equation

  28. Lippert equation - - - - + G v. E _ + + + + 2a

  29. Stokes shifts of naphthalane derivatives ethanol-water solvent mixtures Lakowicz, p. 191

  30. Viscosity sensors

  31. Orientation relaxation of dye solutes(rotational diffusion) Stokes-Einstein-Debye equation f shape factor (spheres f = 1) C friction factor (0<C<1) • local viscosity VM molecular volume T temperaturet k Boltzmann constant

  32. Fluorescence of nile blue on ion exchange resin Habuchi et al., (Sapporo), Anal. Chem. 73, 366-372 (2001) Resin: styrene – divinylbenzene copolymer Cross-linking frequency (): 8 % divinylbenzene Ionexchange group: Na-sulphonate

  33. Determination of or : via measuring fluorescence depolarization

  34. Fluorescence of nileblue adsorbedon ion exchangeresin

  35. Dual fluorescence: twisted intramoleculat charge transfer = TICT Fluoresc. spectrum of DMANCN in ethyleneglycol, the ratio of the intensities of the two bands varies with viscosity Lakowicz, p. 201

  36. Fluorescent amino acids phenyl alanine tyrosine triptophane

  37. Absorption and fluorescence spectra of triptophane (water, pH 7) Lakowicz, p. 446

  38. Fluorescence spectra of tryptophane in different local environments • Apoazurin Pfl • T1 ribonuclease • staphillococcus nuclease • glucagon Lakowicz p. 453

  39. Lakowicz, p. 461

  40. Lakowicz, p. 461

  41. Resonance energy transfer (Förster resonance energy transfer = FRET) Molecular ruler to measure distances! Resolution of optical microscope: max. ~ 200 nm, depends on FRET: detection of 2-10 nm distances

  42. Donor dye – acceptor dye, fluorescence band of D overlaps with absorption band of A

  43. If D and A are close, FRET, exciting D, the energy is transferred to A, fluorescence of A is detectable The FRET effect is proportional to 1/r6

  44. Example for application of FRET: study of DNA –phospholipid interaction C. Madeira, Biophys. J. 85, 3106 (2003)

  45. Acceptor Donor: EtBr (ethidium bromide)

  46. BODIPY fluorescence EtBr absorption

  47. Conformational changes of proteins can be monitored

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