B PRINCIPLES OF FLUORESCENCE SPECTROSCOPY Kasha's rule although Vavilov reported in 1926 that quantum yields were generally independent of excitation wavelength.Upon excitation into higher electronic and vibrational levels,the excess energy is quickly dissipated, 0-2 leaving the fluorophore in the lowest vibrational level of S1.This relaxation occurs in about 1012s and is presum- ably a result of a strong overlap among numerous states of Abs.XEmis nearly cqual energy.Because of this rapid relaxation,emis- sion spectra are usually independent of the excitation DISTANCE WAVELENGTH wavelength.Exceptions exist,such as fluorophores which exist in two ionization states,each of which displays Figure1,8.Mirror inage rule and Franck-Condon factors distinct absorption and emission spectra.also.some mole cules are known to emit from the S2 level,but such emis- sion is rare and ge observed in biological ability (Franck-Condon factor)between the zeroth and erally pot second vibrational levels is largest in absortion the recipro molecules It toask why perylene follows the mio cal transition is also oustest of th st pr pable in em ission image rule.but s that the een in its excitat on sp 3t31 and and emission spectrab approp 335nm( e1.3 nin The clo uldexist b ve orpt ra e(/D and F()/D here e(is th cited state (S). api 1 y to ( extinction co curs predominantly from relative photon flux over a wavenumber increment Ar nce.el inglet state().The emission spectrum of quinine is the Agreement between these spectra is generally found for mirror image of the SoS absorption of quinine,not of polynuclear aromatic hydrocarbons. its total absorption spectrum.This is true for most fluoro phores:the emission is the mirror image of the SoS 1.3.C.Exceptions to the Mirror Image Rule absorption,not of the total absorption spectrum. The generally symmetric nature of these spectra is a Although the mirror image rule often holds,many excep result of the same transitions being involved in both ab- tions to this rule occur.This is illustrated for p-terphenyl sorption and emission and the similarities of the vibra in Figure 1.9.The absorption spectrum of p-terphenyl is tional energy levels of So and S.In many molecules these devoid of structure.but the emission spectrum shows vi- energy levels are not significantly altered by the different brational structure.such deviations from the mirror image electronic distributions of So and S.According to the rule usually indicate a different geometric arrangement of franck-Copdon principle.all electronic transitions are nuclei in the excited state as compared to the ground state vertical.that is.they occur without change in the position Nuclear displacemen tscan occu of the nuclei.As a ult if a partic of the relatively long lif time of the S state e.which allow *o 30 20℃ 20 0.5 A 250 300 350 WAVELENGTH【nm) Figure 1.9. ption(A)and emission (F)spectra of p-tepheny
1.INTRODUCTION TO FLUORESCENCE 9 -1.0F 1.0 Pyrene Excime 360400440480520560 WAVELENGTH (nm) Figure 1.10.Emi ne in t 400450500550 WAVELENGTH【nm) Figure 1.11.Emission spe 3 time for motion following the instantaneous proc duced with permission from John Wiley and Sons, om Ref absorption.In the case of p-terphenyl,it seems likely tha the individual rings become more coplanar in the excited state. As a result,the emission spectrum is more highly structured than the absorption spectrum.In addition to pend on pH(Figure 1.12).The pK for dissociation of the being an exception to the mirror image rule,p-terphenyl is proton is 5.45 for acridine in the ground state.However. unusual in that its emission spectrum shows more vibra- emission from the acridinium group can be observed at tional structure than its absorption spectrum.The opposite higher pH values.This occurs because the pKa of the is generally observed. excited state of acridine is 10.7,and thus acridine can bind Excited-state reactions other than geometric rearrange a proton from the solvent during its excited-state lifetime.1 ments can also result in deviations from the mirror sym- Changes in pKa in the excited state also occur for bio metry rule.One example is shown in Figure 1.10,which chem cal fluorophores.For example,phenol and tyrosine omioeooonmeRhonc each show two emissions the long-wavelength emission being favored by a high conc ntration of proton acceptors wavelengths is a mirror image of the absorption spectrum of the of anthracene.The unstruct red emission at longe wave. in e to 4 in the ited state.Following lengths is due to formation of a charge is lost to between the excited state of anthracene and diethylaniline c proton in the ng The u uctured emission is from this complex Many e phe phe late n ma polynuclear ar matic hydrocarbo s such as and also dominate the on spectrum. with These excited mplexes are Acridine es can also forn m c mp lexes with The best- pyre 49 a highl concentrations the Thi s long-wavelength e可 du to ex term excimer being an abbreviation for an state dime 600 rexcited-state processes can oc ur which shift th WAVELENGTH nm sp ctra and may or may not change the Profile.Acrid
10 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 1.4.FLUORESCENCE LIFETIMES AND ear 10ns.For the fluorophore illustrated in Figure 1.13 QUANTUM YIELDS the lifetime is The fluorescence lifetime and quantum yield are perhaps 1 the most important characteristics of a fluorophore.The t=下+ko 1.21 quantum yield is the number ofemitted photons relative to the number of absorbed photons.Substances with the One should remember that fluorescence emission is a largest quantum vields.approaching unity.such as rho- dom p and fev damines,display the brightest emission.The lifetime is p The lifetime is an ave rage value of ines the time avail. a singl ential the flu ophore to inte ract with diffuse in its (g.[1.13,be w),63%of the molecules have decayed nent,and hence the nformation available from its prior tot=t and 37%decay at>. sion. The lifetime of the fluorophore in the absence of nonra- The meaning of the quantum yield and lifetime is best diative processes is called the intrinsic or natural lifetime and is given by represented by a simplified Jabtonski diagram (Figure 1.13).In this diagram we do not explicitly illustrate the individual relaxation processes leading to the relaxed S tn=T [13 state Instead we focus attention on those processes re. In principle,the natural lifetimecan be calculated from the abs ctra extin fficient,andemiss orophore (T) decay rate rcan tio of the num ber of photons emi number absorbed. The pro F(v)dD (v) esses governed by the rate constants r and knur both T=2.88×109n2 depopulate the excited state.The fraction of fluorophores F(v) which decay through emission,and hence the quantum vield.is given by =28x102%j鸭产 【1.4 Q2下+kw [1.1J where is the emission spe ctrum plotted on the r(cm The quantum yield can be close to unity if the radiationless .is the sorpti and n is the refractive index of the medium.The integrals decay rate is much smaller than the rate of radiative decay are calculated over the So>S absorption and emission that is,k.We note that the energy yield of fluore spectra.In many cases this expression works rather well is alw vays less than unity bec of Stokes'lo particularly for solutions of polynuclear aromatic hydro- carbons.For instance the calculated value of r for tive decay processes with the single rate constant The lifetime of the excited state is defined by the average 容 time the molecule spends in the excited state prior to return ved for perylene which displays aqua eld to the ground state.Generally,fluorescence lifetimes are s wh q.1 fail.Th exp sio es no interaction wit the solvent,do no consider changes in refractive index(n)betwe the ab sorption andemission wavelength,and assumes nochange in excited-state geometry.A more complete form of Eg. [1.4](not shown)includes a factor G=gi/gu on the right- h hand side,where gland gu are the degeneracies of the lower and upper states.respectively.For fluorescence transitions G=1.and for phosphorescence transitions,G= The natural lifetime can be calculated fr om the asured Figure 1.13.Asimplified Jablofski diagram lifetime()and quantum yield:
1.INTRODUCTION TO FLUORESCENCE 11 =VO [1.5]1.4.A.Fluorescence Quenching Equation [1.5]can be derived from Eqs.[1.1]-[1.3].Many The intensity of fuorescence can be decreased by a wide Such d s in inte biochemical fluorophores do not behave as predictably as unsubstituted aromatic compounds.Hence,there is often h poor agreement between the value of t calculated from o Eq.[1.5]and that calculated from the absorption and emission spectra(Eq.[1.4]).These discrepancies occur for L ng a variety of unknown and known reasons,such as a fraction gur of the fluorophores being located next to quenching groups,which sometimes occurs for tryptophan residues in proteins. qu The quantum yield and lifetime can be modified by the pro intensity is describe by the e well-kno factors which affect either of the rate constants (Tor k) Volmer equation. For example,a molecule may be nonfluorescent as a result of a fast rate of internal conversion or a slow rate of emission.Scintillators are generally chosen for their high Fo=1+KTQ]=1+kdolQ] [1.6 quantum yields.These high yields are a result of large I values.Hence,the lifetimes are generally short,near 1 ns. In this expression K is the Stern-Volmer quenching con- The fluorescence emission of aromatic substancescontain stant,ke is the bimolecular quenching constant,to is the ing-NO2 groups is generally weak,primarily as a result unquenched lifetime,and [Q]is the quencher concentra- of large values fork The quantum yields of phosphores- tion.a wide variety of molecules can act as collisional cence are extremely small in fluid solutions at room tem- quenchers.Examples include oxygen,halogens,amines, perature.The triplet-to-singlet transition is forbidden by and electron-deficient molecules like acrylamide.The symmetry,and the rates of spontaneous emission are about mechanism of quenching varies with the fluorophore 10's"or smaller.Since k values are near 10s"quan- quencher pair.For instance,quenching of indole by acry- tum yields of phosphoresceace are small at room tempera- lamide is probably due to electron transfer from indole to ture.From Eq.[1.1]one can predict phosphorescence acrylamide,which does not occur in the ground state. quantum yields of 10-6 Quenching by halogens and heavy atoms occurs due to There are instances where comparison of the natural spin-orbit co upling and intersystem crossing to the triplet lifetime,measured lifetime, and quantum vield can be state (Figure 1.5). informative.For instance in the case of the widely used Besides collisional quenching,fluorescence quenching membrane probe 1,6-diphenyl-1.3.5-hexatriene(DPH)the can occur by a variety of other processes.Fluorophores can measured lifetime near 1o nsis much lo er than that form ponfluorescent comnlexes with calculated from Eg.1.1].which is near 1.5 ns.7 In this case the calculation based on the absor m of the ground state and does not rely on diffusion or molecular DPHis inco rrect because the absorption transition is toa collisions.Quenching can also oc cur by a variety of trivial state of diffe nt electronic sy than the echanisms such as attenuation of the state such a en in light by the fluo rophore itself or other absorbing re mplex fluo hores with he FRET (10-1%s) h k [Q] 10-1s S -A Figure 1.14.Jablofiski diag with collisional o y transfer (FRET).The term Et is used to represent nonradiative paths to the ground state besides quenching and FRET.Revised from Ref.20
12 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 1.4.B.Time Scale of Molecular Processes in Sol This pro s is callec solvent relaxation and occurs in 10 s in fluid The phenomenon of quenching provides a valuable context It is thes differ ces between absorption and emission for understanding the role of the excited-state lifetime in that result in the high sensitivity of emission spectra to allowing f月mo scence measurements to detect dynamic solvent polarity,and the smaller spectral changes seen I Pro cesses in solution or in macromolecules The basic idea Solvent relaxaton can result in sub is that absorption is an instantaneous event.According to shifts.In proteins,tryptophan the Franck-Condon principle,absorption occurs so fast absorb light at 280 nm,and their fluorescenc emission that there is no time for molecular motion during the rs near 350n Although 10 ns may appear to be a absorption process.Absorption occurs in the time it takes brief time span it is in fact quite long relative to the a photon to travel the length of a photon,in less than 10-15 motions of small molecules in fluid solution.In the folle s Hence abso rption sne opy can only vield infor ing section we will explain how dynamic rotational diffu tion on the av age ground state of the molecules which sion can be studied by measuring the polarization or absorb light Only solvent molecules that are immediatelv anisotropy of the emission. adiacent to the absorbing species will affect its absorption nat se sitive to molecu dynamics and can only provide information on the 1.5.FLUORESCENCE ANISOTROPY nt to the chro phore The length of time fluo ain in the Anisotropy m rements are commonly used in the bio excited state rrovides an unity for inte chemical appli ations of fluores py meas other mole les in solution nching of fluo urements prov d nformation on the nd shape o proteins or the rigidity of vario he expansio of time and dista ovided by the fluc me Anisotropy measuremen have 1i atime If a f n the measure protein-protein associa tions and fluidity of the fluc membranes and for imunoassays of numerous substan Anisotropy measurements are bas d on the principle o nt (D)of photoselective excitation of fluorophores by polarized light.Fluorophores preferentially absorb photons whose theygen mole diffuse 108 is given by electric vectors are aligned parallel to the transition mo- quation ment of the fluorophore.The transition moment has a defined orientation with respect to the molecular axes.In △2=2D元 [1.7 an isotropic solution.the fluorophores are oriented ran domly.Upon excitation with polarized light,one selec. The distance is about 70 A,which is comparable to the tively excites those fluorophore molecules whose thickness of a biological membrane or the diameter of a absorption transition dipole is parallel to the electric vector protein.Some fluorophores have lifetimes as long as 400 of the excitation.This selective excitation results in a ns,and hence diffusion of oxygen molecules may be artially oriented population of fluorophores(photoselec- observed over distances of 450A.In contrast,absorption tion)and in partially polarized fluorescence emission. measuremen s are only sensitive to the immediate environ Emission also occurs with the light polarized along a fixed ment around the fluorophore.and then only sensitive to the axis in the fluorophore.The relative angle between these instantaneously averaged environment moments determines the maximum measured anisotropy Other examples of dynamic p cesses in solution in (ro;see Eq.[10.20]).The fluorescence anisotropy(r)and volve fluorophore-solvent interactions and rotational dif- polarization(P)are defined by fusion as was obser ption 【1.8 Most fluorophores have large nents in the r= 1+214 excited state than in the gr d state.Rotational motions of small solvent molecules in fluid solution are rapid urring on a time cale of 40 ps or less.The 【1.9] +11 allows amnle ime for the whereI and I are the fluore ce intensities of the excited-state dipole,which lowers its energy and shifts (1) po