1.INTRODUCTION TO FLUORESCENCE 13 when the sample is excited with vertically polarized light. 1.6.RESONANCE ENERGY TRANSFER Anisotropy and polarization are both expressions for the same phenomenon,and these values can be interconverted Another important process that occurs in the excited state using Eqs.[10.3]and [10.4]. s resonance energy transfer(RET).This process occurs several phenomena can decrease the measured anisot whenever the emission spectrum of a fluorophore,called ropy to values lower than the maximum theoretical values the onor,verapswith thabsortio stmof The most common cause is rotational diffusion.Such other molecule,called the acceptor. Such overlap is diffusion occurs during the lifetime of the excited state and llustrated in Figure 1.15.The acceptor does not need to be displaces the emission dipole of the fluorophore.Measure- fluorescent.It is important to understand that RET does ment of this parameter provides information about the not involve emission of light by the donor.RET is not the relative angular displacement of the fluorophore between result of emission from the donor being absorbed by the the times of absorption and emission.In fluid solution, acceptor.Such reabsorption processes are dependent on most fluorophores rotate extensively in 50-100 ps.Hence, the overall concentration of the acceptor,and on non- the molecules can rotate many times during the 1-to 10-ns molecular factors such as sample sizc,and are thus of less excited-state lifetime,and the orientation of the polarized nterest.There is no intermediate photon in RET.The emission is randomized.For this reason,fluorophores in donor and acceptor are coupled by a dipole-dipole inter- aqueous nonviscous solution typically display anisotropies action.For these reasons,the term RET is preferred to the near zero transfer ofexcitation between fluorophores also term fluorescence resonance energy transfer (FRET), esults in decreased anisotropies which is also in common use. The effects of rotational diffusion can be decreased ifthe The extent of energy transfer is determined by the dis fluorophore is bound to a macromolecule.For instance,it tance between the donor and acceptor and the extent of is known that the rotational correlation time for the protein spectral overlap.For convenience,the spectral overlap human serum albmin (HSA)is near 5o ns sun se HSA (Figure 1.15)is described in terms of the Forster distance s covalently labeled with a fuorophore whose lifetime is (Ro).The rate of energy transfer kr(r)is given by 10 ns.Assuming no other processes result in loss of anisot- ropy,the expected anisotropy is given by the Perrin equa- tion: 【1.11 where r is the distance bet een the donor (D)and the ro 1+(/0 【1.10j acceptor (A),and to is lifet ime of the donor in the absence rgy trans er.The effic ency or e ergy trans fer for a single donor-acceptor pair at a fixed distance is where ro is the anisotrop of ro which would be measured in the and is the rotational E= R+5 [1.12 ne for the diffusio In this din of the th A d to 033 s tha WAVELENGTH Figure 1.15.Spectral overlap for nuc ergy trans fer (RET)
14 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY Hence,the extent of transfer depends on distance(r). Fortunately,the Forster distances are comparable in size to biological macromolecules,30-60 A.For this reason, energy transferhas been used as a"spectroscopic ruler"for measurements of distance between sites on proteins.The value of Ro for energy transfer should not be confused with Wavelength (nm) Time (ns the fund The complex.The theory 0.4 rent for donors tha are coval linked,free in solution,or contained in the restricted geometries of membranes or DNA.Additionally,depend ing on the donor lifetime,diffusion can increase the extent of energy transfer beyond that predicted by Eq.[1.12]. Time (minutes) Time (ns) 1.7.STEADY-STATE AND TIME-RESOLVED FLUORESCENCE Figure1.16. Fluorescence measurements can be broadly classified into two types of measurements.steady-state and time-resolved. where lo and mo are respectively.the intensities and anisot ents ar erformed with con This is the att-d in stant illumination and obs theexcitation ased to illustr mon type of mea The sample is ill minated with the de nes what can be obse a continuous beam of light,and the intensity or emission The steady-state tropy(r)is given by spectrum is recorded (Figure 1.16).Because of the the average ofr(weighted by I(): nanosecond timescale of fluorescence,most measure- ments are steady-state measurements.When the sample is first exposed tolight.steady state is reached almost imme iately r(t)I(t)dt 1.15 The second type of mea ements,time resolved meas r=0 urem ts,isus uringinte nsity de ays or ani J1o血 ropy decays.For thes measurements,the sample is exposed to a pulse of light.where the pulse width is typically shorter than the decay time of the sample(Figure In this equation the der nominator 1.16).This intensity decay is recorded with a high-speed detection system that permits the intensity or anisotropyto erator the s to the be me sured on the n dy-state an ity at time stand that ther arather sim stitution of Eqs.[1.13]and [1.14]intoEq.[1.15]yields nship bet teady state and time-resolved meas the Perrin equation (Eq.[1.10]). urements.The steady-state o ervation is simply an average Perhaps a simpler example is how the steady-state inten of the time-resolved phenomena over the intensity decay of sity Iss is related to the decay time.The steady-state the sample.For instance,consider a fluorophore which intensity is given by displays a single decay time (and a single rotational correlation time(0).The intensity and anisotropy decays lss=∫loe-trt=lor [1.16 are given by 0 I()=loe/ u.13lTievaincofoeantbecoasitcredoeapaantrntc neter which nds on the f of rop rs He r(=roe [1.14 steady- tate sity is propor the lifeti e Thi
1.INTRODUCTION TO HUORESCENCE 15 makes sense in consideration of Eqs.[1.1]and [1.2],which near 280 nm and emits near 340 nm.The emission spec- showed that the quantum yield was proportional to the trum of indole is highly sensitive to solvent polarity.The lifetime. emission of indole may be blue-shifted if the group is buried within a native protein(n),and its emission may 1.7.A.Why Time-Resolved Measurements? shift to longer wavelengths(red shift)when the protein is Whereas steady-state flu cence measurements are sim unfolded (u). ple,nanosecond time-resolve me rements typically re Membranes typically do not display intrinsic fluores- quire complex and expensive instrumentation.Given the cence.For this reason,it is common to label membranes relationship between the steady-state and time-resolved with probes which spontaneously partition into the nonpo- measurements,what is the value of these more complex lar side-chain region of the membranes.One of the most measurements?It turns out that much of the molecular commonly used membrane probes is DPH.Because of its information available from fluorescence is lost during the low solubility and quenched emission in water,DPHemis- sion is seen only from membrane-bound DPH.Other lipid time-averaging process.For example,anisotropy decays of fluorescent macromolecules are frequently more com- probes include fluorophores attached to lipid or fatty acid plex than a single exponential (Eq.[1.14)).The precise chains,as shown for rhodamine B in Figure 1.17. shape of the anisotropy decay contains information about Although DNA contains nitrogenous bases which look like fluoro the shape of the macromolecule and its fexibility.Unfor- tnately this shape information is lost during raging of the anisot ver the decay time ([1)Im ously to DNA such as acridines,ethidium bromide,and other planar cationic species.For this reason,staining of d cells with dyes that bind to DNA is widely used to visualize anis iple, and identify chromosomes.There are a few naturally oc the ic.In pra not uffic al the curring fluorescent bases,such as the Y-base,which oc curs in the anticodon region of a phenylalanine tRNA h (Figure 1.17.bottom). ys al mation that is lost A wide variety of other substances display significant during the averaging pro ently,macror cence.among biological molecules one can ob can exist in more than a single conformatic ,and the de ay fluorescence from reduced nicotina mide adenine time of a bound probe may depend on conformation.The din otide (NADH),from oxidized flavins (FAD,the intensity decay could reveal two decay times.and thus the leotide).and presence of more than one conformational state. The idoral phosphate as wel 1 as fr chlor L steady-state intensity will only reveal an average intensity ally. of inte seent or is dependent on a weighted average of the two decay times not flu t in a ient of the UV-visible There are pumerous additional reasons for measuring of extr have h time-resolved fluorescence.In the presence of energy ed for labelin the transfer.the intensity decays reveal how acceptors are T. ost widely dan ylchloride(DNS-Cl distributed in space around the donors.Time-resolved ands for h ents re veal whether quenching is due to diffu sion or to tion with the pd-state fuc gure 1.18.Th In en much the mole la. in pro ich content is available only from time-resolved (DN or gree obtai esired flu sign ul m ays 1.8.BIOCHEMICAL FLUOROPHORES of the luorcphoresaredividediniotwog ple,a neral classes,intrinsi whicl added 2 to a sampl h es not display the desired properties.In proteins, indole group of tryptophan (Figure 1.17).Indole absorbs can expe h
16 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY RhB.fatty acid ester 00 500 300 400500 600 WAVELENGTH Inm) Flgure 1.17.Absorption and emis enmission from witew-yCH STM from Ref 20 monophospate (AMP)(Figure 1.18).but it may be too large to fit into some binding sites or in a DNA helix.It DNS-CI fluorescent probe is one that displays a high intensity,is stable during continued illumination,and does not sub- stantially perturb the biomolecule or process being studied 1.8.A.Fluorescent Indicators Another class of fluorophores consists of the fluorescent indicators.These are fluorophores whose spectral proper- OH ties are sensitive to a substance of interest.One example is PBFI,which is shown in Figure 1.19.This fluorophore E-ATP lin-benzo-AMP contains a central azacrown ether,which binds K'.Upon K+binding,the emission intensity of PBFI increas AMP鬥 allowing the amount of K'to be determined.Flu orescent
1.INTRODUCTION TO FLUORESCENCE 17 1.0 TNS-Ape s 4NH4 Absorptior Emission TNS in DMPC PBFI +K TNS n H2O 400 450 500 550 WAVELENGTH(nm】 Figure 1.20.Emis ion spectra of TNS in water,bound to ap 60.5 -.c because emission spectra are sensitive to the fluomo no K* phore's environment.the spectra of extrinsic probes are oftenused to determine a probe's location on a macromole- 300 400 500 600 cule.for example.one of the widely used probes for such WAVELENGTH (nm) studies is 6-(-toluidinyDn (TNS)(Fig 10),which aphthalene-2sulfonic additional favor indicators are presently available for wide variety substances.including Ca Mg Na?C and O biomo for pH.The tion of fluc escence to che mical sens other widely used probes.including the DNA stair ethidium bromide.The protein apomyoglobin contains a hydrophobic pocket which binds the heme group.This pocket can also bind other nonpolar molecules.Upon the 1.9 MOLECULAR INFORMATION addition of apomyoglobin to a solution of TNS,there is a FROM FLUORESCENCE large increase in fluorescence intensity,as well as a shift of the emission spectrum to shorter wavelengths.This Emission Spectra and the Stokes' increase in TNS fluorescence reflects the nonpolar charac- membranes (F e1.20).Thc The most dramatic aspect of fluorescence is itsoc of TNS bound tha hich ah GC wh phospha is sor h The shifts. lar flu hores in pol compared The ore nd its im e group of tryptophan resic n pr the membrane are more polar.From the emission spectrum I the it appears that TNS binds to the polar head-group tra of in the location or uyp region of the membranes,rather than to the nonpolar proteins. acyl side-chain region.Hence,the emission spectra of due (longer wav solvent-sensitive fluorophores provide information inthe protei on the location of the binding sites on the macromole- cules. the a tryptophan ng of a 1.9.B.Quenching of Fluorescence residue is As described in Section 1.4.A,a wide eules or ions can act as quenchers of fluorescence: