Principle of fluorescence
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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


Outline

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

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


Fluorescence

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


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

π-bond


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

fluorescein


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


Quenching

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.


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

    solution.

d


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.

Retinol


Extrinsic fluorphores

Extrinsic fluorphores

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

fluorescence

wavelength


Extrinsic fluorphores1

Extrinsic fluorphores

  • Different Stoke’s shift of

    rhodamine derivatives.

wavelength

1.Fluorescein

2.Rhodamine 6G

3.Tetramethylrhodamine

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

    transfection.


Principle of fluorescence

GFP


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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