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