FluorescenceSpectrophotometry
Peter TC So,
Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
Chen Y Dong,
Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
Fluorescencespectrophotometry isaclassoftechniquesthat assaythe stateofabiologicalsystem by studying its interactions with fluorescent probe molecules. This interaction ismonitored by measuring the changes in the fluorescent probe optical properties.
The Electronic Excited State
Fluorescence and phosphorescence are photon emissionprocesses that occur during molecular relaxation fromelectronic excited states. These photonic processes involvetransitions between electronic and vibrational states of polyatomic fluorescent molecules (fluorophores). TheJablonski diagram (
Figure 1
) offers a convenient represen-tation of the excited state structure and the relevanttransitions. Electronic states are typically separated byenergiesontheorderof10000cm
2
1
.Eachelectronicstateis split into multiple sublevels representing the vibrationalmodes of the molecule. The energies of the vibrationallevels are separated by about 100cm
2
1
. Photons withenergies in the ultraviolet to the blue-green region of thespectrum are needed to trigger an electronic transition.Further, since the energy gap between the excited andground electronic states is significantly larger than thethermal energy, thermodynamics predicts that moleculepredominately reside in the electronic ground state.The electronic excited states of a polyatomic moleculecan be further classified based on their multiplicity. Theindistinguishability of electrons and the Pauli exclusionprinciple require the electronic wave functions to haveeithersymmetricorasymmetricspinstates.Thesymmetricwave functions, also called the triple state, have threeforms, multiplicity of three. The antisymmetric wavefunction, also called the singlet state, has one form,multiplicity of one.To the first order, optical transition couples states withthe same multiplicity. Optical transition excites themoleculesfromthelowestvibrationalleveloftheelectronicground state to an accessible vibrational level in an ele-ctronic excited state. Since the ground electronic state is asingletstate,thedestinationelectronicstateisalsoasinglet.After excitation, the molecule is quickly relaxed to thelowest vibrational level of the excited electronic state. Thisrapid vibrational relaxation process occurs on the timescale of femtoseconds to picoseconds. This relaxationprocess is responsible for the Stoke shift. The Stoke shiftdescribes the observation that fluorescence photons arelonger in wavelength than the excitation radiation.The fluorophore remains in the lowest vibrational levelof the excited electronic state for a period on the order of nanoseconds, the fluorescence lifetime. Fluorescenceemission occurs as the fluorophore decay from the singletelectronicexcitedstatestoanallowablevibrationallevelinthe electronic ground state.Thefluorescenceabsorptionandemissionspectrareflectthe vibrational level structures in the ground and theexcited electronic states, respectively. The Frank–Condonprinciple states the fact that the vibrational levels are notsignificantly altered during electronic transitions. Thesimilarity of the vibrational level structures in the groundand excited electronicstates often resultsin theabsorptionand emission spectra having mirrored features.Theelectronicexcitedstatealsohasspecificpolarizationproperties. Fluorophores are preferentially excited whenthe polarization of light is aligned along a specificmolecular axis (the excitation dipole). Further, thefluorescencephotonssubsequentlyemittedbythemoleculewill have polarization orientated along another molecularaxis (the emission dipole). In general, the excitation andemission dipoles do not coincide.
Article Contents
Introductory article
.
The Electronic Excited State
.
Radiative and Nonradiative Decay Pathways
.
Factors Affecting Fluorescence Intensity
.
Phosphorescence
.
Instrumentation for Fluorescence Spectrophotometry
.
Applications of Fluorescencein theStudy of BiologicalStructure and Function
S2E S1Internal conversionS0FluoresenceExcitationPhosphorescenceIntersystem crossingT1
Figure1
TheJablonskidiagramoffluorophoreexcitation,radiativedecayand nonradiative decay pathways. E denotes the energy scale; S0 is theground singlet electronic state; S1 and S2 are the successively higher energyexcitedsingletelectronicstates.T1isthelowestenergytripletstate.
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Radiative and Nonradiative DecayPathways
Radiative decay describes molecular deexcitation pro-cesses accompanied by photon emission. Molecules in theexcited electronic states can also relax by nonradiativeprocesses where excitation energy is not converted intophotons but are dissipated by thermal processes such asvibrationalrelaxationandcollisionalquenching.Let
G
and
k
betheradiativeandnonradiativedecayratesrespectivelyand
N
be the fraction of fluorophore in the excited state.The temporal evolution of the excited state can bedescribed by:
d dt
À
À
N
¼
N
0
e
Àð
À
þ
k
Þ
¼
N
0
e
À
=
À
½
2
The fluorescence lifetime,
t
, of the fluorophore measuresthe combined rate of the radiative and nonradiativepathways:
À
3
In the absence of nonradiative decay processes, one candefine the intrinsic lifetime of the fluorophore:
À
4
The ‘efficiency’ of the fluorophore can then be quantifiedby the fluorescence quantum yield,
Q
:
ÀÀ
5
FactorsAffectingFluorescenceIntensity
A number of factors contributes to the nonradiative decaypathways of the fluorophores and reduces fluorescenceintensity. In general, the nonradiative decay processes canbe classified as:
k
5
k
ic
1
k
ec
1
k
is
[6]where
k
ic
isthe rateof internalconversion,
k
ec
istherate of external conversion, and
k
is
is the rate of intersystemcrossing.Internal conversion is a process where the electronicenergy is converted to the vibrational energy of thefluorophore itself. Since vibrational processes are drivenbythermalprocesses,theinternalconversionratetypicallyincreases with temperature, which accounts for thecommonlyobserveddecreaseinfluorescenceintensitywithrising temperature.External conversion describes the process where thefluorophore loses electronic energy to its environmentthroughcollisionwithothersolutes.Collisionalquenchingprocesses are particularly interesting as they allow thebiochemical environment of the fluorophores to bemeasured. A number of important solute molecules, suchas oxygen, are efficient fluorescence quenchers. Uponcollision, the fluorophore is deexcited nonradiatively. Thecollisional quenching rate can be expressed as:
k
ec
5
k
0
[
Q
] [7]where
k
0
isrelatedtothediffusivityandthehydrodynamicsradii of the reactants and [
Q
] is the concentration of thequencher.When collisional quenching is the dominant nonradia-tive process, eqn [1] predicts that fluorescence lifetimedecreases with quencher concentration:
8
The steady state fluorescence intensity,
F
, also diminishesrelative to the fluorescence intensity in the absence of quencher,
F
0
. This effect is described by the Stern–Volmerequation:
9
Fluorescence signal reduction can also result from groundstate processes – steady state quenching. A fluorophorecan be chemically bound to a quencher to form a ‘darkcomplex’ – aproductthatdoesnotfluoresce.Fluorescenceintensity decreases with steady state quenching as:
K
where
s
is the association constant of the quencher andthe fluorophore. Fluorescence lifetime is not affected bysteady state quenching as the excited states are notinvolved.
Phosphorescence
Intersystemcrossingisanotherprocesswherefluorescencesignal is reduced and phosphorescence is generated. Spin-orbit coupling is a quantum mechanical process that isresponsible for intersystem crossing. Intersystem crossingdescribes the relaxation of the molecule from a singletexcited state to a lower energy, triplet excitation state.Since spin-orbit coupling is a weak effect, the intersystemcrossingrate islow.Therelaxationfromthetripletstate tothe singlet ground state requires another change of multiplicity. Hence, the decay from the triplet states alsohas a very low rate. However, radiative relaxation,phosphorescence, does occur due to spin-orbit coupling.The typical phosphorescence lifetime is on the order of
Fluorescence Spectrophotometry2
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microseconds to seconds. Phosphorescence has largerStoke shift than fluorescence owing to the triple statehaving lower energy.Since phosphorescence rate is often much lower thanthermally activated nonradiative decay processes such ascollisional quenching, phosphorescence is rarely observedin aqueous systems at physiological temperature. How-ever, a number of protein conformation studies atcryogenic temperatures have utilized phosphorescencespectroscopy.
Instrumentation for FluorescenceSpectrophotometry
The measurement of fluorescence signals provides asensitive method of monitoring the biochemical environ-ment of a fluorophore. Instruments have been designed tomeasure fluorescence intensity, spectrum, lifetime andpolarization.Fluorescenceintensitymeasurementallowsthedetermi-nation of the presence of fluorophores and their concen-trations. Fluorescence intensity measurement is used innumerous biochemical assays. The instrument designs forthese assaysare rather straightforward but are as varied asthe applications.Fluorometersaregeneral-purposeinstrumentsdesignedto measure fluorescence spectrum, polarization and/orlifetime. A typical fluorometer includes a light source, aspecimen chamber with integrated optical components,and highsensitivitydetectors(
Figure2
).The mostcommonlight source for fluorometers are lamp sources, such asxenonarclamps.Theselampsprovidearelativelyuniformintensityoverabroadspectralrangefromtheultraviolettothe near infrared. The optical paths of the excitation andthedetectionlightpathsarealongtheorthogonalaxis.Theorthogonal arrangement ensures minimal leakage of excitation light into the detection side. High sensitivityphotodetectors such as photomultipliers or charge-coupled device cameras are commonly used.For spectral measurement, monochrometers or band-pass filters are placed in the excitation and emission lightpaths to select a specific spectral band. The excitationspectrumisdefinedasthefluorescentintensitymeasuredasa function of excitation wavelength at a constant emissionwavelength; the emission spectrum is the fluorescentintensity measured as a function of emission wavelengthat a constant excitation wavelength.Fluorometers have also been designed to measurefluorescence lifetime. Given the typical lifetime of fluor-ophores, accurate lifetime measurement requires photo-detectors and signal processing electronics withsubnanosecond resolution. High-precision fluorometershave been designed in both the time and frequencydomains. In the time domain, femtosecond or picosecondpulsed lasers are often used as excitation light sources. Atime-correlated single photon counting method is fre-quently employed, in which the time delay between theexcitationlightpulseandtheresultantfluorescencephotonis measured by high-speed electronics. In the frequencydomain,thespecimenisexcitedbyalightsourcewithhigh-frequencycontent.Theresultantfluorescencesignalisalsomodulated at the same frequency but is phase-delayed andamplitude-demodulated. The phase delay and demodula-tion contain the lifetime information of the fluorophoreand can be measured using heterodyning or homodyningdetection techniques.For polarization measurement, polarizers are insertedinto the excitation and emission light paths. With theexcitation polarizer fixed, the emission polarizater can berotated to measure the perpendicular (
I
\
) and parallel (
I
6
)components of the fluorescence emission. The steady statepolarization is defined as:
P
c
À
k
c
k
and an equivalent measure is the steady state anisotropy:
r
c
À
k
c
k
LSEXO SCEMODET
Figure 2
A typical fluorometer design. LS is the light source, EXO is theexcitation optical train, SC is the specimen chamber, EMO is the emissionoptical train, and DET is the optical detector. Both the excitation andemission optical trains contain beam-shaping and collimation optics. For wavelength-resolved measurements, spectral selection opticalcomponents such as monochrometers and filters are included in the EXOand EMO. For polarizationmeasurement, polarizersare added to EXOandEMO.Forlifetimemeasurement,alaserlightsourceisoftenusedandhigh-speed electronics are integrated into the detector subsystem.
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Fluorescence Spectrophotometry
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