PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 19.Fluorescence Sensing 20.1.A.Anisotropy Properties of Metal-Ligand Complexes. .575 19.1.Optical Clinical Cl emistry 531 20.2.Spectral Properties of Metal-Ligand Probes.576 19.2.Spectral Observables for Fluorescence 20.2.A.The Energy-Gap Law 577 sing. 532 20.3.Biophysical Applications of Metal-Ligand 19.2.A.Optical Properties of Tissues. 534 Probes 511 19.2.B.Lifetime-Based Sensing. 534 20.3.A.DNA Dynamics with Metal-Ligand l9.3.Mechanisms of Sensing··· 535 578 19.4.Sensing by Collisional Quenching 536 20.3B.Domain-omain Motions in 19.4.A.Oxygen Sensing g36 S80 19.4.B.Lifetime-Based Sensing of Oxygen 527 20.3.C MLC Lipid Probes. 19.4.C.Mechanism of Oxygen Selectivity 537 20.4.MLC Immunoassays. 38 194n Other ox sors 20.4.A.Long-Wavelength Immunoassays. 585 19.4.E ygen Se 20.4.B.Lifetime Immunoassays Based on 19.4F Oth Quenchers RET 586 19.5. Energy-Tran sing 541 20.5.Clinical Chemistry with Metal-Ligand 195.A. pH and pCO2 Sensing by Encrgy Complexes 587 591 Transfer 541 20.6.Perspective on Metal-Ligand Probes 19.5.B.Glucose Sensing by Energy Transfer References 591 542 19.5.C.Ion Sensing by Energy Transfer 543 Problems 593 19.5.D.Theory for Energy-Transfer Sensing 545 19.6.Two-State pH Sensors 5A5 19.6.A.Optical Detection of Blood Gases 545 21.DNA Technology 197 toind on-Transt r(PET) Anion Sensors 551 21.1.DNA Sequencing 595 19.8. of Analyte Re ognition 552 21 1 A Nucleo 598 19.8.A.Specificity of Cation Probes 。 552 21.1.B Energy-Transfer Dyes for DNA 19.8.B.Theory of Analyte Recognition 598 Sensing. 553 equencing with NIR Probes. 601 198.c.Sodium and Potassium Probes 554 21.1.D.DNA Sequencing Based on Lifetimes 604 19.8.D.Calcium and Magnesium Probes 556 212.High-Sensitivity DNA Stains.········ 604 19.8.E Glucose Probes 559 21.2.A.High-Affinity Bis DNA Stains 605 19.9.Immunoassays 560 21.2.B.Energy-Transfer DNA Stains 606 19.9.A.Enzyme-Linked Im 21.2.C.DNA Fragment Sizing by Flow ssays(ELISA 560 19.9.B Time-Resolved Immunoassays . 560 21.3.DNA Hy 607 607 19.9.C.Energy-Transfer Immunoassays 562 21.3.A DNA Hybridiz ion Measure 19.9.D.Fluorescence Polarization Immunoassays 563 DNA Probe .608 19.10.Conclusions 565 21.3.B.DNA Hybridization Measured by References 56的 Excimer Formation 610 Problems 572 21.3.C.Polarization Hybridization Assays 610 21.3.D.Polymerase Chain Reaction 612 21.3.E.DNA【 agers 612 20.Long-Lifetime Metal-Ligand 21.3.F Light 6 Complexes 21.4.Fluorescenc ed DNA Probe Arrays Hybridiz 613 21.5. erspectives nces 20.1.Introduction to Metal-Ligand Probes Problems
CONTENTS xx研 22.Phase-Sensitive and Phase-Resolved Appendix Il.Fluorescent Lifetime Emission Spectra Standards 1.Nanosecond Lifetime Standards .645 22.1.Theory of Phase-Sensitive Detection of 2. Picosecond lifetime Standards 646 Hluorescence 619 Re sentative Frequency-Domain Intensity 22.1.A.Phase-Sensitive Emission Spectra of cavs 646 a Two-Component Mixture .621 nain Lifetime Standards 647 22.1.B.Phase Suppression. .623 650 22.1.C.Examples of PSDF and Phase 624 22.1.D.High-Fre ency or Low-Prequency Det 2.a-Modulation t ect 26 Appendix III.Additional Reading 1.Lifetime Measurements Spectra 627 .653 2.Which Molecules are Fluorescent 22.2.A.Resolution Based on Phase or Re esentative Emission Spe ctra.and Modulation Lifetimes.·· 627 Practical Advice 654 22.2.B.Resolution Based on Phase Angles scence and Photophysics 654 and modulations 627 Principles of Fluores ce Spect 654 22.2.C.Resolution of Emission Spectra from Bioch nical Fluo 654 Phase and modulation spectra 628 6 、u 655 22.3.Fluorescence Lifetime Imaging Microscopy 620 22.3.A.Phase-Suppressi aging 621 K5< References 63 656 Problems 635 656 656 Sensing 656 13 656 ions of Fluores ence 5 Appendix I.Corrected Emission Spectra Infrared and NIR Fluorescence 5 16 Lasers 17. 7 1.B-Carboline Derivatives as Fluorescence scence Microscopy 18. Metal-Ligand Complexes and Unusual Standards Lmn0 phores,.。++,￥ 657 2.Corrected Emission Spectra of 9,10-Diphenylanthracene,Quinine Sulfate, and Fluorescein 638 3 Long-Wavelength Standards 639 Answers to Problems 659 aviolet standards 639 5 Additional Corrected Emission Spectra. References 643 Index············, 679
Principles of Fluorescence Spectroscopy Second Edition
Introduction to 1 Fluorescence During the past 15 years there has be nthe use offluorescence in the Just a few years ago,fluorescence spectroscopy and time o the groun d state is spin-alowed and curs rapidly by resolved fluorescence were primarily research tools in biochemistry and biophysics.This situation has changed so that fluorescence is now used in environmental moni- is near 10 ns(10x 10s).As will be described in Chapter toring,clinical chemistry,DNA sequencing,and genetic 4.the lifetime ()of a fluorophore is the average time analysis by fluorescence in situ hybridization (FISH),to between its excitation and its return to the ground state.It name afew areas ofapplication.Additionally,fluorescence is valuable to consider a 1-ns lifetime within the context of isused for cellidentification and sorting in flow cytometry, the speed of light.Light travels 30 cm or about one foot in and in cellular imaging to reveal the localization and one nanosecond.Many fluorophores display subnanosec- movement ofintracellular substances by means of fluores- ond lifetimes.Because of the short timescale of fluores- cence microscopy.Because of the sensitivity of fluores- cence,measurement of the time-resolved emission cence detection,and the expense and difficulties of requires sophisticated optics and electronics.In spite of the handling radioactive substances.there is a continuing de- experimental difficulties,time-resolved fluorescence is velopment of medical tests based on the phenomenon of widely practiced because of the increased information fluorescence.These tests include the widely used enzyme available from the data,as compared with stationary or linked immunoassays (ELISA)and fluorescence polariza- steady-state measurements. tion imm Phosphorescenceisemission oflight from tripletexcited While there is continued growth in the applications of states,in which the electron in the excited orbital has the fluorescence,and continued dev same spin orientation as the ground-state electron.Transi- and fluorescent p nain ogy,the re tions to the ground state are forbidden and the emission the and need to h rates are slow (103-100 s-1),so that phosphorescence ence,th the pasic phe lifetimes are typically milliseconds to seconds.Even can longer lifetimes are possible,as is seen from"glow-in-the used d applied rese sures to light,the ph out the book,we have includ examples and applications hat illustrate the principles of fluorescence. hors slowlyr cence urn to he und state.Phos perature. This is bec st many 1.1.PHENOMENON OF ses which compete with FLUORESCENCE hosphore Lamoosbatanc ance ctal-ligan and o .Lumines one cence display mixed t states. These MLCS and phosphorescence depending on excited state.In excited singlet states,the 1
2 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY more rapid phenomenon of fluorescence.However,be- cause of the importance of these new metal-ligand lumi nophores,which bridge the gap between fluorescence and tobe se phosphorescence,their properties are described in Chapter strong daylight or sunshine,but with no cross lights. Fluorescence typically occurs from aromatic molecules or any strong reflected light from behind.If we look Some typical fluorescent substances(fluorophores)are the int shown in Figure 1.1.One widely encountered fluorophore e glass thr is quininc,which is present in tonic water.If one observes a glass of tonic water which is exposed to sunlight,a faint lively blue, blue glow is frequently visible at the surface.This glow is If the liquid be poured out into another vessel,the most apparent when the glass is observed at a right angle cending stream gleams undu relative to the direction of the sunlight and when the n the same ively yet delicat dielectric constant is decreased by adding less polar sol- vents like alcohols.The quinine present in the tonic is in pro this singular nhenemenon excited by the ultraviolet (UV)light from the sun.Upon return to the ground state,the quinine emits blue light with The thinnest film of the liquid seems quite as effec a wavelength near 450 nm.The dependence of the bright- tive in producing this superficial colour as a consider ness of quinine fluorescence on solvent polarity provides able thickness.For instance,if in pouring it from on glass int .the end of the unn be made a noninvasive means of learning more about our neighbors we The first observation of fluorescence from a quinine solu- tion in sunlight was reported by Sir John Frederick William surface.the intensity of the uch that it it Herschel in 1845.The following is an excerpt from this almost impossible to avoid supposing that we have a early report. highly colored liquid under our view. On a Case of Superficial Colour presented by a homo As a footnote,Herschel wrote"I write from recollection geneous liquid internally colourless.By Sir John of an experiment made nearly twenty years ago,and which Frederick Willia Hersche Philosophical Transac I cannot repeat for want of a specimen of the wood." 145 s of the Royal Society of London (1845)135:143 It is evident from this early description that Herschel Received January 28,1845,Read February 3.1845 recognized the presence of an unusual phenomenon which could not be explained by the scientific knowledge of the time.To this day,the fluorescence of quinine remains one The sulphate of e is well k如l o be or own of the most used and most beautiful examples of fluores- cence.However,it is unlikely that Herschel's experiment. weights ofthe sulnhate and of crvstallizedar described from memory 20 vears later.would be acceptec rubbed up together with addition of a very little water by the Patent Office.Herschel (Figure 1.2)was from a dissolve entirely and immediaely.It is this solution distinguished family of scientists who lived in England but largely diluted,which exhibits the opticalphenomenon had their roots in Germany.2 For most of his life,Herschel h2emThoehprtceodytanaspareantandoloi did research in astronomy,publishing only a few nl,or a eresting to notice that the first known fuor ertain incidences of the light an sible for the d beautiful celestial blue colourwhich,from the circum the fure ers,which ap stances of its occurrence,would seem to originate in 1950 World W3 II the t of De he light titst s in entering d i thd and ing ng a whyzing the lud ne Thi those assay Health to very small depthwithin the tid 3 pectr 0P0 ed-or are encount d in daily life i To see the colour in question o advantage.all that is ang cen in a n equal propor esp ctively (Figur Polynu aromatic hydrocar