2Outline Luminescence : fluorescence or phosphorescence? Jablonski diagramCharacteristics of fluorescence emissionFluorescence lifetime and quantum yieldQuantum mechanic behindQuenchingBeer-Lambert lawBiochemical fluorescence
3phosphorescencePhosphorescence – 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 :
4FluorescenceFluorescence – electron go back to ground state from singlet excited state. Fluorescence has emission rate about 108 s-1 (lifetime≈ns).Pic. From :
5Jablonski 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)Absorption10-15IC10-12ISC10-8FP
8Exception 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…etfluorescein
9Emission 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
11Fluorescence 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.
12Quantum 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.
13Quantum yield of some fluorephores Quantum Yield [Q.Y.] StandardsQ.Y.[%]Conditions for Q.Y. MeasurementExcitation [nm]Cy34PBS540Cy527620Cresyl Violet53Methanol580Fluorescein950.1 M NaOH, 22°C496POPOP97Cyclohexane300Quinine Sulfate580.1 M H2SO4, 22°C350Rhodamine 101100Ethanol,25°C450Rhodamine 6G94Ethanol488Rhodamine B31Water514Tryptophan13Water, 20°C280L-Tyrosine14275
14QuenchingEnery 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.
15Collisional quenching Oxygen, halogen, amines, and electron-deficient molecule often act as quenchers.In simplest quenching, stern-volmer equation holdsKD is stern-volmer quenching constant, kq is bimolecular constant, τ0 is unquenched lifetime.
16Static quenching Energy is taken by forming complex. Combine with collisional quenching
17Modify Stern-Volmer plots Some of fluorphores are accessibile and some aren’t for quenchers.
18Time 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 equationAbout 7 nm. Concentration of quenching would process isIn 25 oC water, oxygen dissolve is about 10-4 M
19Optical densityIn optics, density is the transmittance of an optical element for a given length and a given wavelength.In fluorescence, opticaldensity indicates us theabsorption of fluorescentsolution.d
20Beer-Lambert lawAbsorption 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 lawwhere c is concentration (M) and ε is the extinction coefficient (M-1cm-1)
21Extinction coefficient Extinction 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
22Inner 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 formulaUsually, solution with OD<0.05 avoids IFE.
23Biochemical fluorophores Intrinsic fluorphoresExtrinsic fluorphoresDNA probesChemical sensing probesFluorscent protein