Principle of fluorescence
OutlineLuminescence : fluorescence or phosphorescence?Jablonski diagramCharacteristics of fluorescence emissionFluorescence lifetime and quantum yieldQuantum mechanic behindQuenchingBeer-Lambert lawBiochemical fluorescence
phosphorescencePhosphorescence electron go back to ground state from triplet excited state (which is forbidden). Thus, it has lower rate about 103~100 s-1 (life timems~s)
Pic. from : http://www.glassner.com/andrew/cg/research/fluphos/fluphos.htm
FluorescenceFluorescence electron go back to ground state from singlet excited state. Fluorescence has emission rate about 108 s-1 (lifetimens).
Pic. From : http://en.wikipedia.org/wiki/Image:Fluorescent_minerals_hg.jpg
Jablonski diagramJablonski 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)Absorption10-15IC10-12ISC10-8F10-8P10-3-100
Exception of mirror imageRelaxation time is much smaller than emission, E is much bigger than emission.Excimer excited state dimer.Influence of solvent pH, O2et
Emission characteristic Stokes shiftObviouly, 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 Stokes shift, usually shown in diagram by wavelength or wavenumber difference.
Q1 and Q0 are energies of vibration taken by surround molecules.Q1Q0
Fluorescence lifetimeFirst order rate equation!
Unfortunately, theres also contained nonradiative decay in nature.
ko-1 is called natural lifetime, (ko+knr)-1 is real lifetime.
Quantum yieldDefinition 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 [Q.Y.] StandardsQ.Y.[%]Conditions for Q.Y. MeasurementExcitation [nm]Cy34PBS540Cy527PBS620Cresyl Violet53Methanol580Fluorescein950.1 M NaOH, 22C496POPOP97Cyclohexane300Quinine Sulfate580.1 M H2SO4, 22C350Rhodamine 101100Ethanol,25C450Rhodamine 6G94Ethanol488Rhodamine B31Water514Tryptophan13Water, 20C280L-Tyrosine14Water275
QuenchingEnery 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 quenchingOxygen, 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, 0 is unquenched lifetime.
Static quenchingEnergy is taken by forming complex.
Combine with collisional quenching
Modify Stern-Volmer plotsSome of fluorphores are accessibile and some arent for quenchers.
Time scale of molecular processes in solutionIs 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 densityIn optics, density is the transmittance of an optical element for a given length and a given wavelength.
In fluorescence, opticaldensity indicates us the absorption of fluorescentsolution.
Beer-Lambert lawAbsorption of light go through a substance is proportional to the effective cross section(), concentration of molecules(n) and intensity(I).
Rewrite the Beer-Lambert law
where c is concentration (M) and is the extinction coefficient (M-1cm-1)
Extinction coefficientExtinction coefficient is calibrated by a fluorescent solution with width 1 cm and concentration 1 mole per liter.
fluorphoresExtinction coefficient (M-1cm-1)Rhodamine 6G105,000Rhodamine B123,000Cell tracker (BLue)16,000SYTOX38,000
Inner filter effect (IFE)Solution with optical density absorbs not only excitation light but also emission light.Excitation IEFEmission IEF absorbs by solute or fluorphoresCorrection of IFE could be wrote down in the following formula
Usually, solution with OD
Biochemical fluorophoresIntrinsic fluorphoresExtrinsic fluorphoresDNA probesChemical sensing probesFluorscent protein
Intrinsic fluorphoresVitamine A Retinol, in liver stellate cell and retina. Retinol
Extrinsic fluorphoresEg. FITC, rhodamine conjugate with protein, dextran, antibodyetc. for labeling specific target.wavelengthfluorescence
Extrinsic fluorphoresDifferent Stokes shift of rhodamine derivatives.
wavelength1.Fluorescein2.Rhodamine 6G3.Tetramethylrhodamine4.Lissamine rhodamine B5.Texas Red
DNA probesHoechst33342 (binding to minor groove of DNA) Red: rhodamine dextran blue: hoechst33342
Fluorescent proteinGFP Green fluorescence proteinExtracted from jellyfishAequorea victoria.Vector contained DNA of GFP is used in celltransfection.
As a reporterGFP vectorPut in liposomePlace into cells by injection or fusion
Use as NFkB reporterEGFP vector