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Principle of fluorescence

Principle of fluorescence. Outline. Luminescence : fluorescence or phosphorescence? Jablonski diagram Characteristics of fluorescence emission Fluorescence lifetime and quantum yield Quantum mechanic behind Quenching Beer-Lambert law Biochemical fluorescence. phosphorescence.

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Principle of fluorescence

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  1. Principle of fluorescence

  2. Outline • Luminescence : fluorescence or phosphorescence? • Jablonski diagram • Characteristics of fluorescence emission • Fluorescence lifetime and quantum yield • Quantum mechanic behind • Quenching • Beer-Lambert law • Biochemical fluorescence

  3. phosphorescence • Phosphorescence – electron go back to ground state from triplet excited state (which is forbidden). Thus, it has lower rate about 103~100 s-1 (life time≈ms~s) Pic. from : http://www.glassner.com/andrew/cg/research/fluphos/fluphos.htm

  4. Fluorescence • Fluorescence – electron go back to ground state from singlet excited state. Fluorescence has emission rate about 108 s-1 (lifetime≈ns). Pic. From : http://en.wikipedia.org/wiki/Image:Fluorescent_minerals_hg.jpg

  5. Jablonski diagram • Jablonski diagram can schematically tell us the fluorescence activity. It is proposed by Professor Alexander Jablonski in 1935 to describe absorption and emission of light.

  6. π-bond

  7. Mirror image

  8. Exception of mirror image • Relaxation time is much smaller than emission, ΔE is much bigger than emission. • Excimer – excited state dimer. • Influence of solvent – pH, O2…et fluorescein

  9. Emission characteristic – Stoke’s shift • Obviouly, form the Jablonski diagram of previous page, we know energy of emission light is less than energy of absorption light. • This energy shift is called “Stoke’s shift”, usually shown in diagram by wavelength or wavenumber difference. • Q1 and Q0 are energies of vibration taken by surround molecules.Q1≈Q0

  10. Stoke’s shift

  11. Fluorescence lifetime • First order rate equation! • Unfortunately, there’s also contained nonradiative decay in nature. • ko-1 is called natural lifetime, (ko+knr)-1 is real lifetime.

  12. Quantum yield • Definition is is the ration of the number of photons emitted to the number of photons absorbed. That is emission efficiency. • Quantum yield can be calculated from standard quantum yield.

  13. Quantum yield of some fluorephores

  14. Quenching • Enery of excited state could be taken by other substance, this process is called fluorescence quenching. • Collisional (dynamics) quenching and static (complex- forming) quenching are most often process in quenching.

  15. Collisional quenching • Oxygen, halogen, amines, and electron-deficient molecule often act as quenchers. • In simplest quenching, stern-volmer equation holds KD is stern-volmer quenching constant, kq is bimolecular constant, τ0 is unquenched lifetime.

  16. Static quenching • Energy is taken by forming complex. • Combine with collisional quenching

  17. Modify Stern-Volmer plots • Some of fluorphores are accessibile and some aren’t for quenchers.

  18. Time scale of molecular processes in solution • Is quenching rapidly happened? • Ex. quenching by O2, which has diffusion coefficient 2.5 x 10-5 cm2/s. The average distance of an O2 can diffuse in 10ns is given by Eistein equation • About 7 nm. Concentration of quenching would process is • In 25 oC water, oxygen dissolve is about 10-4 M

  19. Optical density • In optics, density is the transmittance of an optical element for a given length and a given wavelength. • In fluorescence, optical density indicates us the absorption of fluorescent solution. d

  20. Beer-Lambert law • Absorption of light go through a substance is proportional to the effective cross section(σ), concentration of molecules(n) and intensity(I). • Rewrite the Beer-Lambert law where c is concentration (M) and ε is the extinction coefficient (M-1cm-1)

  21. Extinction coefficient • Extinction coefficient is calibrated by a fluorescent solution with width 1 cm and concentration 1 mole per liter.

  22. Inner filter effect (IFE) • Solution with optical density absorbs not only excitation light but also emission light. • Excitation IEF • Emission IEF – absorbs by solute or fluorphores • Correction of IFE could be wrote down in the following formula • Usually, solution with OD<0.05 avoids IFE.

  23. Biochemical fluorophores • Intrinsic fluorphores • Extrinsic fluorphores • DNA probes • Chemical sensing probes • Fluorscent protein

  24. Intrinsic fluorphores

  25. Intrinsic fluorphores • Vitamine A – Retinol, in liver stellate cell and retina. Retinol

  26. Extrinsic fluorphores • Eg. FITC, rhodamine – conjugate with protein, dextran, antibody…etc. for labeling specific target. fluorescence wavelength

  27. Extrinsic fluorphores • Different Stoke’s shift of rhodamine derivatives. wavelength 1.Fluorescein 2.Rhodamine 6G 3.Tetramethylrhodamine 4.Lissamine rhodamine B 5.Texas Red

  28. DNA probes • Hoechst33342 (binding to minor groove of DNA) Red: rhodamine dextran blue: hoechst33342

  29. Fluorescent protein • GFP – Green fluorescence protein • Extracted from jellyfish Aequorea victoria. • Vector contained DNA of GFP is used in cell transfection.

  30. GFP

  31. As a reporter • GFP vector • Put in liposome • Place into cells by injection or fusion • Use as NFkB reporter EGFP vector

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