1.INTRODUCTION TO FLUORESCENCE c2-5 %"tiewn CH O H Quinine Fluorescein Rhodamine B og。 7-Hydroxy- POPOP Acridine Orange coumarin or Umbelliferone Figure 1.1.Structures of typical fluorescent substances. bons,such as anthracene and perylene,are also fluores cent,and the emission from such sp ecies is used for r envi ronmental monitoring of oil pollution.Some substitu ted rganic compounds are also fluorescent.for exa 14-bis(5-phenvloxazol-2-yl)benzene (POPOP)is ed in scintillation counting,and Acridine is ofte use as a DNA stain.Cou marins are also highly fluor are often used as fluoroge enic probes in enz uch ase nzyme-linkedi nunosorbent assays (ELISA).In this case the rent molecule is ty umbellifer phosphate,.which is。 of the h xylphosphate by e phospha t s in ous add examples co ould be pre sented.In of listin he bo ppe wit dividual of fu used n8 00 th ides orb are shielo from the olvent by highe filled orbitals.The lanthanides display long decay times because of this shielding.and they have low emission rates because of their small extinction coef ficients. Fluorescence spectral data are generally presented as emission spectra.A fluorescence emission spectrum is Flgure 1.2.Sir John Frederick Wiiam Herschel( plot of the fluorescence intensity versus wavelength 1,1871).Repre (nanome rs)or wavenumber (cm Royal Socicty of Chemistry
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY WAVELENGTH (nm) 350370 Absorpti WAVELENGTH (nm) 10 Absorption /Emission 5500315002750023509 WAVENUMBER (cm-1) Flgure 1.3. ngths are shown for convenience.See 1.2.IABLONSKI DIAGRAM The processes which occur between the absorption and widely and are dependent u emission of light are usually illustrated by a Jablofiskis a orn eov diagram.Jablofiski diagrams are often used as the starting structure ption and emission.The dissolved.The spectra of some compounds,such as ariety of form toi arious mole perylene,show significant structure due to the individual vibrational energy levels of the ground state and excited processes whic an ccur in excited state .The states.Other compounds,such as quinine,show spectra grams are named after Professor Alexander Jablofski(Fig- ure 1.4),who is regarded as the father of fluorescence whichare devoid of vibrationls spectroscopy because of his many accomplishments,in- t featu is high-sensitivity dee6on-TmcesiviyofnUoIecGncewsedia187 cluding his descriptions of concentration depolarization to demonstrate that the rivers Danube and Rhine were am学oe connected by underground streams>This conpection was demonstrated by placing fluorescein (figure 11)into the Brief History of Alexander Jabtonski Danube river so me 60hours later, Professor Jablonski was born February 26,1898,in n a small river which led to the Rhine.Toay fuorescein is still used as eme Voskresenovka,Ukraine.In 1916 he began his study of atomic physics at the University of Kharkov.His marker for locating individuals at sea,as has been seen on study was interrupted by military service first in the the landing of space capsules in the Atlantic Ocean.Read- Russian Army and later in the newly organized Polish ers interested in the history of fluorescence are referred to Army during world War l At the end of 1918.when the excellent summary by Berlman.5 an indeper ndent Poland was recreated after more than
1.INTRODUCTION TO FLUORESCENCE 5 120 years of occupation by neighboring powers great energy organized the Department of Physics, Jablonski left Kharkov and arrived in Warsaw,where which became a scientific center for studies in atomic he entered the University of Warsaw to continue his and molecular physics. study of physics.His study in Warsaw was again His work continued past his retirement in 1968 interrupted in 1920 by his military service during the Professor Jablonski created a spectroscopic school of Polish-Bolshevik war. thought which persists even today through his numer- An enthusiastic musician,Jablorski played the first ous students who now occupy positions at universities violin at the Warsaw Opera from 1921 to 1926parallel in Poland and elsewhere.Professor Jabloriski died on to his studies at the university under Stefan Pienkowski. September 9,1980.More complete accounts of He received his doctorate in 1930 for work "On the Jablonski's accomplishments are given in Refs.7 and 8. influence of the change of wavelengths of excitation light on the fluorescence spectra."Although Jabionski A typical Jablonski diagram is shown in Figure 1.5.The left the Warsaw Opera in 1926 and devoted himself singlet ground,first,and second electronic states are de entirely to scientific work,music remained his great picted by So.S.and S2.respectively.At each of these passion until the last days of his life. electronic energy levels the fluorophores can exist in a Throughout the 1920s and 1930s the Department of number of vibrational energy levels,denoted by0,1.2.etc. Experimental Physics at the University of Warsaw was In this diagram we have excluded a number of interactions an active center for studies on luminescence under which will be discussed in subsequent chapters,such as S.Pienkowski.During most of this period,Jablonski quenching,energy transfer,and solvent interactions.The worked both theoretically and experimentally on fun transitions between states are depicted as vertical lines to damental problems of photoluminescence of liquid illustrate the instantaneous nature of light absorption solutions as well as on the effects of pressure on atomic Transitions occur in about 10-1s s,a time too short for spectral lines in gases.The problem that intrigued significant displacement of nuclei.This is the Franck- Jablonski for many years was the polarization of pho- Condon principle. toluminescence of solutions The energy spacing between the various vibrational mental facts,he distinguished the transition moments energy levels is illustrated by the emission spe tum of in absorption and in emission and analyzed various perylene (Figure 1.3).The individual en ission maxim fa ctors respo onsible for the depolarization of lumines. rgy le els)are about 1500 cm cence apart.At room temperature.the lablorski's work was interrupted once rgy is not ad pulate the excited d in A bs ule ith the lo the polish army and he sp of fire the germany ar and ther 6 rmy in 1946 nce het citad is tool he retu ned to Poland to chair a new de mal nd it is for this sics in the new Nicholas Co icus University in light and Torun.This beginning occured in the ver heat to ind y diffi Following light abso e eral sse ally destr spite ational level of eithe some highe With a few rare excep sion Absorption hv Phosphorescence Figure 1.5. One form of a Jablofiski diagram
6 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY tions,molecules in conde d phases rapidly relax to the are infrequent.Generally,if any of the characteristics lowest vibrational level of S.This proces s is called inter described in the following sections are not displayed by a nal conversion and generally occurs in 10- s or less.SInce given fluorophore,one may infer some special behavior fluorescence lifetimes are typically near 10s,internal for this compound. conversion is generally complete prior to emission.Hence, fluorescence emission generally results from a thermally 1.3.A.Stokes'Shift equilibrated excited state,that is,the lowest-energy vibra- Examination of the Jablonski diagram(Figure 1.5)reveals tional state of S. that the energy of the emission is ty ically less than that of return to the ground state typically occurs to a higher ion Hence flu ically occur at lo excited vibrational ground-state level,which then quickly ies or long This (10s)reaches thermal equilibrium (Figure 1.5).An first observed b Sir G.G.Stokes in 1852 interesting consequence of emission to higher vibrational ents used relatively si ground states is that the emission spectrum is typically a gure 1.6).The mirror image of the absorption spe ctrum of the s S vided by s lass filte ich as par transition.This similarity occurs because electronic exci ed glas y tation does not greatly alter the nuclear ge ne ight belo 00 which s abs rbed h inine (Fis cinn of the vi n re 1 3)The light Ae a the r(eye)b ay 8 on and th d i n It is nteres ting to read Stokes'des sioatohefirsturiple go a spi conver ion of his obser T m T is term wing paragraph is fro his report publish no: horescence and is ge d to longer wave engths (lowe ergy)relative to the fluores nce.Con version calledintersystemcrossing.Transition On the from Ti to the singlet ground state is forbidden,and,as a of Pe result,rate constants for triplet emission is several orders in the Uni of magnitude smaller than those for fluorescence.Mole ofCa cules containing heavy atoms such as bromine and iodine Society of London (852)142:463-562. Received May i.read may 27 1852 are frequently phosphorescent.The heavy atoms facilitate intersystem crossing and thus enhance phosphorescence quantum yields. The following originated inaconsider d by 13.CHARACTERISTICS OF described by him intwo papers printed in the Philo FLUORESCENCE EMISSION sophical Tr sactions for 1845 entitled"on a case of Colour presented by a The phenomenon of fluorescence displays a number of general characteristics.Exceptions are known,but these to he ranspare at8onm eieiongs glass of wine) Transmits 400 nm A G.G.Stokes Figure 1.6. ofthe Stokes'shif
1.INTRODUCTION TO FLUORESCENCE 7 water,when viewed by transmitted light,exhibits nev- ble for the blue glow of lower refrangibility or fre- erthele ss in cert ain aspects,and unde certain inci quency.Thus,invisible UV rays were absorbed to ences the light,a produce the blue light at the surface.Stokes later f Sir John Her suggested the use of optical properties.such as absorp mall but finite thickness adiacent to the surface hy tion,colored reflection,and fluorescence,to identify which the light enters.After passing through this stra- organic substances. tum,the incident lighi,though not sensibly enfeebled Later in life,Stokes was universally honored with nor colored,has lost the power of producing the same degrees and medals.He was kni ghted in 1889 and re ma e co ame Master of Pembroke Colle in 1902.After the 1850s,Stokes bec nini and his tra om Careful reading paragraphre eral im kes diedon February lutioniscolb S( the UV which we 1.3.B.Emission Spectra Are Typically near the surfac This is be Independent of the Excitation Wavelength elatvely conc and absorb all of the n the Another general property of fluorescence is that the same first several millimeters.Hence.Stok s ot ed the inner fluorescence emission spectrum is gencrally obse rved ir filter effect.After passing through the solut the light espective of the excit wav e ength.This is kn was"enfeebled"and no longer capable of causing the blue glow.This occurred because the UV was remove d and the 'enfeebled"light could no longer excite quinine.However, had Stokes used a second solution of fluorescein,rather than quinine,it would have still been excited because of the longer absorption wavelength of fluorescein. Energy losses between excitation and emission are ob served universally for fluorescent molecules in solution. One common cause of the stokes'shift is the ranid decav to the lowest vibrational level of s.Furthermore.fluoro phores generally decay to higher vibrational levels of So (Figure 15),resulting in further loss of excitation nalization of the excess vibrat In these can displ shifts e to sol ects, excited ate rea energy transfer Brief History of Sir G.G.Stokes Professor Stokes (Figure 1.7)was born in Ireland, August 3,1819.He entered Pembroke College,Cam- bridge,in 1837 and was elected as a fellow of Pem- broke College immediately upon his graduation in 1841.In 1849 Stokes became Lucasian Professor at Cambridge a chair once held by Newton.Because of ent for the e chair,he also worked in the olved wi th vide of scientific incl ng ynami city the aready known w he ente ridg n Profes paper on he unde st light of a higher"refrangibility"or frequency was responsi Centre,Royal Sociery of Chemistry