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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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Principle of fluorescence

Principle of fluorescence



  • 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 – 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 :



  • Fluorescence – electron go back to ground state from singlet excited state. Fluorescence has emission rate about 108 s-1 (lifetime≈ns).

Pic. From :

Jablonski diagram

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.

Principle of fluorescence


Mirror image

Mirror image

Exception of mirror image

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


Emission characteristic stoke s shift

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

Stoke s shift

Stoke’s shift

Fluorescence lifetime

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.

Quantum yield

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.

Quantum yield of some fluorephores

Quantum yield of some fluorephores



  • 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.

Collisional quenching

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.

Static quenching

Static quenching

  • Energy is taken by forming complex.

  • Combine with

    collisional quenching

Modify stern volmer plots

Modify Stern-Volmer plots

  • Some of fluorphores are accessibile and some aren’t for quenchers.

Time scale of molecular processes in solution

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

Optical density

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



Beer lambert law

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)

Extinction coefficient

Extinction coefficient

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

Inner filter effect ife

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.

Biochemical fluorophores

Biochemical fluorophores

  • Intrinsic fluorphores

  • Extrinsic fluorphores

  • DNA probes

  • Chemical sensing probes

  • Fluorscent protein

Intrinsic fluorphores

Intrinsic fluorphores

Intrinsic fluorphores1

Intrinsic fluorphores

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


Extrinsic fluorphores

Extrinsic fluorphores

  • Eg. FITC, rhodamine – conjugate with protein, dextran, antibody…etc. for labeling specific target.



Extrinsic fluorphores1

Extrinsic fluorphores

  • Different Stoke’s shift of

    rhodamine derivatives.



2.Rhodamine 6G


4.Lissamine rhodamine B

5.Texas Red

Dna probes

DNA probes

  • Hoechst33342 (binding to minor groove of DNA)

Red: rhodamine dextran blue: hoechst33342

Fluorescent protein

Fluorescent protein

  • GFP – Green fluorescence protein

  • Extracted from jellyfish

    Aequorea victoria.

  • Vector contained DNA

    of GFP is used in cell


Principle of fluorescence


As a reporter

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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