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Principle of fluorescence. Outline Luminescence : fluorescence or phosphorescence? Jablonski diagram Characteristics of fluorescence emission Fluorescence.

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Presentation on theme: "Principle of fluorescence. Outline Luminescence : fluorescence or phosphorescence? Jablonski diagram Characteristics of fluorescence emission Fluorescence."— Presentation transcript:

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 10 3 ~10 0 s -1 (life time≈ms~s) Pic. from :

4 Fluorescence Fluorescence – electron go back to ground state from singlet excited state. Fluorescence has emission rate about 10 8 s -1 (lifetime≈ns). Pic. From : 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. Time scale (s) Absorption10 -15 IC10 -12 ISC10 -8 F P10 -3 -10 0

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, O 2… 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. Q 1 and Q 0 are energies of vibration taken by surround molecules.Q 1 ≈Q 0

10 Stoke’s shift

11 Fluorescence lifetime First order rate equation! Unfortunately, there’s also contained nonradiative decay in nature. k o -1 is called natural lifetime, (k o +k nr ) -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 Quantum Yield [Q.Y.] Standards Q.Y.[%]Conditions for Q.Y. Measurement Excitation [nm] Cy34PBS540 Cy527PBS620 Cresyl Violet53Methanol580 Fluorescein950.1 M NaOH, 22°C496 POPOP97Cyclohexane300 Quinine Sulfate580.1 M H 2 SO 4, 22°C350 Rhodamine 101100Ethanol,25°C450 Rhodamine 6G94Ethanol488 Rhodamine B31Water514 Tryptophan13Water, 20°C280 L-Tyrosine14Water275

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 K D is stern-volmer quenching constant, k q is bimolecular constant, τ 0 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 O 2, which has diffusion coefficient 2.5 x 10 -5 cm 2 /s. The average distance of an O 2 can diffuse in 10ns is given by Eistein equation About 7 nm. Concentration of quenching would process is In 25 o C 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 -1 cm -1 )

21 Extinction coefficient Extinction coefficient is calibrated by a fluorescent solution with width 1 cm and concentration 1 mole per liter. fluorphoresExtinction coefficient (M -1 cm -1 ) Rhodamine 6G105,000 Rhodamine B123,000 Cell tracker (BLue)16,000 SYTOX38,000

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 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. wavelength fluorescence

27 Extrinsic fluorphores Different Stoke’s shift of rhodamine derivatives. wavelength 1.Fluorescein2.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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