18 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY that is,they decrease the intensity of the emission.These 1.9.C.Fluorescence Polarization or substances include iodide (I),oxygen,and acrylamide. Anisotropy The accessibility of fluorophores to such quenchers can be As described in Section 1.5,fluorophores absorb light used to determine the location of probes on macromole- along a pat lar dit with molecula cules or the porosity of proteins and membranes to guench- axes.F ple,DPHab ers.This concept is illustrated in Figure 1.21,which shows ts long a bs only d along gure 1.17) ligh pohich a fluor the emission intensity of a protein-or membrane-bound phor tes during the excit e lifetime det rmine fluprophore in the odide.As s pres cfOfhewaiersolublequ its polarizat n on the right-hand side of the fig on or anisotropy.The phenomenon of fluores cence polarization can be used to measure the apparent the emi n intensity of a tryptopha on the protein's volume (or molecular weight)of proteins.This measure face (W2),or on the surface of a cell membrane (P2), ment is possible because larger proteins rotate more will be decreased in the presence of a water-soluble slowly.Hence,if a protein binds to another protein,the quencher.The intensity of a buried tryptophan residue rotational rate decreases,and the anisotropy(s)increases (Wi)or of a probe in the membrane interior(P1)will be (Figure 1.22).The rotational rate of a molecule is often less affected by the dissolved iodide,as seen on the left- described by its rotational correlation time 0,which is hand side of the figure.Alternatively,one can add lipid- given by soluble quenchers,such as brominated fatty acids,to study the interio acy side-chain egion s thr meast rements of of quenching by the lipid- 0-=器 [1.17刀 quencher. where n is the viscosity,Vis the molecular volume,Ris Trp (W the gas constant,and T is the temperature in Kelvins. Suppose a protein is labeled with DNS-CI (Figure 1.22. middle).If the protein associates with another protein,the Protein Tryptopha Surface Tryptophan Saturated fatty acids or Prob or Probe Monomer Aggregate 入(nm) A【nm1 W1 or P1 Unsaturated Lipids Saturated Lipids W2 or P2 [a] [o] Figure 1.21.Acce Figure 1.22.Fluc snriation,prolein association,and mem printed, sity.From Ref.20
1.INTRODUCTION TO FLUORESCENCE 19 volume increases and so does the rotational correlation ing to Eq.[1.12],the distance between a donor and ac- time.This causes the anisotropy to increase because of the ceptor can be calculated from the transfer efficiency. relationship between the steady-state anisotropy r and the The use of RET to measure protein association and rotational rrelation time (Fa 11.101 distance is shown in Figure 1.23 for two monomers which Fluorescence polarization measuren ents have also been associate to form a dimer.Suppose one monomer contains to det rminethe e ppa sity of the side chair ophan(trp)residue,and the other a dansyl group. region(center)of men of the trp donor emission with the dansyl acceptor absor probe like DPH (Figure 1.22,bottom),which partitions into the membrane.The viscosity of membranes is known the inte sity of the to decrease in the presence of unsaturated fatty acid side ent quenching can be used to chains.Hence,an increase in the amount of unsaturated or-t the dim g.[1.12]).tis fatty acid is expected to decrease the anisotropy.The also import 。tha RET vides ethod t ure prot curs eve mpingthepolarizaionofthepobehd red in the the door and acceptor are within the Forster distance with observed in utions of known vis otropy measure nents e widely used in bioch 1.10.FLUORESCENCE SENSING istry and are even used for clini cal imi ssays.On reason for this use is the ease with which these absolute In addition to the use of in biochemistry tin values can be measured and compared between laborato- se for analy The us ries. clinical diagnostics is part of th continu way打 1.9.D.Resonance Energy Transfer the use of radioactive tr e use op al me eliminates the dangers of handling radioactive materials Resonance energy transfer(RET rescence resonance enery tranefede and the cost of their proper disposal. Fluorescence is used for a wide variety of biomedical opportunity to measure the distances between sites on purposes.Fluorescence imaging of gels is used to detect macromolecules.Forster distances are typically in the DNA fragments following electrophoretic separation.The range of 15-60 A,which is comparable to the diameter of newer DNA stains provide high detection sensitivity and many proteins and to the thickness of membranes.Accord- can mostly replace the use of 32P and autoradiography.The Donor Acceptor Dimer Trp DNS rp DNS Monomers ner 生1.0 4000 0.5 2000 -M 300 400 500 WAVELENGTH nm Figure 1.23.Energy transfer between donor (D)and acceptor(A)-labeled monomers,which associate to form dimer.In this case the donor is tryptophan and the acceptor is a dansyl group (DNS)
20 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY tional and ranslational diffusion,formation of comees with solvents or solutes,and reorientation of the environ ment surrounding the altered dipole moment of the excited pH state.These dynamic processes can affect the fluorescence Glucose Detector anisotropies,quantum yields,lifetimes,and emission Ca spectra.In addition,resonance energy transfer provides a pCO2 reliable indicator of molecular proximity on the angstrom Sensor- size scale.As aresult,the spectral characteristics of fluoro phores can provide a great deal of information on the Filter 1 solution behavior of macromolecules. LED Light Fluorescence spectroscopy offers the advantages of sen Source sitivity,simplicity,and a wealth of molecular information. Fluorescence measurements are highly sensitive because the observations are made against a dark background.The advantage of a dark background is illustrated by our ability Figure 1.24.Fuoresence sening in clinical cbemistry to see stars at night,but not in the daytime when the sky is bright.While instrumentation for time-resolved fluores- cence is often complex,instruments for steady-state fluo expanding use of DNA sequencing and the Human rescence are relatively simple.This is why there is Cm3peo时o currently a proliferation of instruments for microscopic imaging and macroscopic imaging of gels and multiwell nameniac5r电ntciohoanR plates.Thesensitivity of fluorescenceto numerous cations, anions,or nearby acceptors has resulted in the extensive Figure 1.19 are known for a wide variety of analytes, including most of the ionic species in blood.Fluorescence assays have also been described for immunochemical de- there is a rather direct connection between the spectral tection of a wide variety of low-molecular-weight drugs observables and molecular features of and for highe to visualize how the spectral pr perties are affected by the local enviro nment,accessibility to quenchers.or the pres are also While ence of nearby acceptors.It is this connection between what the molecules are doing and how this affects the e sampl nay be emitted light which allows he design orescent sens ng chemistry ca within the syringe or capillary tube into which the sample scientific qu is drawn,or on the side of the container.The sensor is read by a low-cost fluorescence instrument,which may use REFERENCES light-emitting diodes or laser diodes (laser pointers)as the light source.Within the next decade,one can anticipate the introduction of numerous point-of-care fluorescence as- by a ho says for use at the bedside,in the doctor's office,or for home health care. Gillispic,C.C.(ed.).197"John Frederick William Herschel."in Undenfriend.1995.Develop 1.11.SUMMARY inSd4:542-55 4 。R The essence of any experiment is the existence of an .12:181-209 rman,1.B.1971.Han dbook of Fluo e Spectru ofAromatic observable quantity and the correlation of the value of this observable yAcaomeaoaofinteresLTheimespa 6. en between the abs ion of light and its von Farbst nZP%y94:38-46 sion alloy tim fo process ch of ctral obs 8. Polska Akademia Naul k Instytut Fizyki,1978.Acta Physica Polo mca.Europhys.J.A65(6)
1INTRODUCTION TO HLUORESCENCE 21 9.Stokes.G.G1852.On the change of refrangibility of light Phil. PROBLEMS c transitions in com- 1. Estir 1. plex mo les,Disc.Faraday Soc.9:14-19. .B9 Photophysics Mo 12.Re.,p.108 13.Lakowicz.J.R.and Balter.A.1982.Analysis of excited stat andphosphorescence states,but the emissiverates(T)vary greatly.Emission spectra,lifetimes ()and quantum -modulation fluorescence spectroscopy, yields(0)for eosin and erythrosin B(Er B)are shown in 14.Gafni A Figure 1.25 tions of acridine studied by nanosecond fluoromtry.hem Phys. A. 15. nic Molecutar Photophysics, osin and for Er R B Which rate accounts for the lower quantum yield of 16.Stickler,S.and Berg.R.A962.Relationship between absorp Er B? tionineynduorChemy Phosphorescence lifetin are typically near 1-10 81482 ms.As ne tha he nat e Tor phosp on of ns and th re the let state as for the singlet state Estimate the phosphorescence quantum yields of eo 20.koie2.1R,199s.H0 ence sp copy of biomolecules sin and Er B at room temperature. in Encyclopedia of Molecular Biology and Molecular Medicine,R. d New 12 Estim d 1013、28U a mhoenhate with in e (Problem 1.1) estimate the a 647. tum yield of the 5 state 22 1.3 Thermal population of vibrational levels:The emission spectrum of perylene (Figure L.3)shows equally spaced 298 2.le BremeC.KramerScmi Webet eaks wh and Senior.A.E.994.lin-Boons-ATP and-ADP:Versatile fluo- rescent pro emical studies,J.Flud 24. 1.4 Anisorropy ofa Labeled Protein:Naphthylamine sulfonic Connell.C.R.Heiner.C.Kent,S.B.H.and Hood,L.E.1986. acids are widely used as extrinsic labels of proteins cence detection in automated DNA sequenc analysis,Na A number of derivatives are available.One little know 321:674-679 1Dam.R.J.Hobbs,F.W.Robe but particularly useful derivative is 2-diethylamino-5 C w K.1987.A system for rapid DNA sequencing with fluorescent 1.0 Eosin/ 6 .1997 980.SPIE.Bellinghan 品 ra618 27.Lakowicz,J.R.(ed),1994.Topics in Fluorescence Spectroscopy. Votume4.Probe Design and Chemical Sensing.Plenum Press,New aM 1960 Paths of motecular exeitation Radiat Res.2:243. Erythrosin B 75 9.LakowiczJ.R.and Weher,G.W.1973. by oxygen A cules 550 600 30.Hagag.N.Bimbaum.E.R.and Darall.D.W.1983.Resonance WAVELENGTH (nm) en transfer between cysteine-34.trypto phan-214,and tyrosine 411 of hnman serum albumin,Biochemistry 22:420-2427. Figure1.25
22 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 2000 DENS in 0.1 M Phosphate Buffer 1.0 pH7.0 150 1000 0.5 350400450500550600 WAVELENGTH (nm) Figure 1.26.Absorption and emission spectraof DENS.The quantum yield relative to that of quinine sulfate is84,and its lifetime is near 30 ns naphthalenesulfonicacid(DENS),whichdisplays a life-1.5.Effect of Distance on the Efficieney of FRET:Assume the time near 30 ns,29 longer than that of most analogous presence of a single donor and acceptor and that the onhtheT7280ndemtneanoiDGSs distance between them(r)can be varied. A A oesaomaemcDEwSa0a9 ofDENSiso 30 B.What is the transfer efficiency when the donorandhe correlation time of 30 ns.What is the anisotropy? acceptor are separated by 0.5Ro.Ro.and 2R? B.Assume now that the protein is bound toan antibody 0.7 HSA Anthranilate 0.6 2N 0.5 4, 1.0 0.4 0.3 0.2 o.1 1 260 340 420 R/Ro WAVELENGTH (nm