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Principle of fluorescence

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Presentation on theme: "Principle of 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 103~100 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 108 s-1 (lifetime≈ns). Pic. From :

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) Absorption 10-15 IC 10-12 ISC 10-8 F P

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
Quantum Yield [Q.Y.] Standards Q.Y.[%] Conditions for Q.Y. Measurement Excitation [nm] Cy3 4 PBS 540 Cy5 27 620 Cresyl Violet 53 Methanol 580 Fluorescein 95 0.1 M NaOH, 22°C 496 POPOP 97 Cyclohexane 300 Quinine Sulfate 58 0.1 M H2SO4, 22°C 350 Rhodamine 101 100 Ethanol,25°C 450 Rhodamine 6G 94 Ethanol 488 Rhodamine B 31 Water 514 Tryptophan 13 Water, 20°C 280 L-Tyrosine 14 275

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. fluorphores Extinction coefficient (M-1cm-1) Rhodamine 6G 105,000 Rhodamine B 123,000 Cell tracker (BLue) 16,000 SYTOX 38,000

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 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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