Fluorescence Spectrophotometry Is a Class of Techniques That Assay the State of a Biological Instrumentation for Fluorescence Spectrophotometry

Fluorescence Introductory article Spectrophotometry Article Contents . The Electronic Excited State Peter TC So, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA . Radiative and Nonradiative Decay Pathways Chen Y Dong, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA . Factors Affecting Fluorescence Intensity . Phosphorescence . Fluorescence spectrophotometry is a class of techniques that assay the state of a biological Instrumentation for Fluorescence Spectrophotometry . Applications of Fluorescence in the Study of Biological system by studying its interactions with fluorescent probe molecules. This interaction is Structure and Function monitored by measuring the changes in the fluorescent probe optical properties.

The Electronic Excited State indistinguishability of electrons and the Pauli exclusion Fluorescence and phosphorescence are photon emission principle require the electronic wave functions to have processes that occur during molecular relaxation from either symmetric or asymmetric spin states. The symmetric electronic excited states. These photonic processes involve wave functions, also called the triple state, have three transitions between electronic and vibrational states of forms, multiplicity of three. The antisymmetric wave polyatomic fluorescent molecules (fluorophores). The function, also called the singlet state, has one form, Jablonski diagram (Figure 1) offers a convenient represen- multiplicity of one. tation of the excited state structure and the relevant To the first order, optical transition couples states with transitions. Electronic states are typically separated by the same multiplicity. Optical transition excites the energies on the order of 10 000 cm 2 1. Each electronic state molecules from the lowest vibrational level of the electronic is split into multiple sublevels representing the vibrational ground state to an accessible vibrational level in an ele- modes of the molecule. The energies of the vibrational ctronic excited state. Since the ground electronic state is a levels are separated by about 100 cm 2 1. Photons with singlet state, the destination electronic state is also a singlet. energies in the ultraviolet to the blue-green region of the After excitation, the molecule is quickly relaxed to the spectrum are needed to trigger an electronic transition. lowest vibrational level of the excited electronic state. This Further, since the energy gap between the excited and rapid vibrational relaxation process occurs on the time ground electronic states is significantly larger than the scale of femtoseconds to picoseconds. This relaxation thermal energy, thermodynamics predicts that molecule process is responsible for the Stoke shift. The Stoke shift predominately reside in the electronic ground state. describes the observation that fluorescence photons are The electronic excited states of a polyatomic molecule longer in wavelength than the excitation radiation. can be further classified based on their multiplicity. The The fluorophore remains in the lowest vibrational level of the excited electronic state for a period on the order of nanoseconds, the fluorescence lifetime. Fluorescence emission occurs as the fluorophore decay from the singlet electronic excited states to an allowable vibrational level in S2 the electronic ground state. The fluorescence absorption and emission spectra reflect the vibrational level structures in the ground and the excited electronic states, respectively. The Frank–Condon principle states the fact that the vibrational levels are not Internal conversion significantly altered during electronic transitions. The E S1 Intersystem crossing similarity of the vibrational level structures in the ground and excited electronic states often results in the absorption and emission spectra having mirrored features. Excitation Fluoresence T1 The electronic excited state also has specific polarization properties. Fluorophores are preferentially excited when S0 the polarization of light is aligned along a specific Phosphorescence molecular axis (the excitation dipole). Further, the fluorescence photons subsequently emitted by the molecule Figure 1 The Jablonski diagram of fluorophore excitation, radiative decay and nonradiative decay pathways. E denotes the energy scale; S0 is the will have polarization orientated along another molecular ground singlet electronic state; S1 and S2 are the successively higher axis (the emission dipole). In general, the excitation and energy excited singlet electronic states. T1 is the lowest energy triplet state. emission dipoles do not coincide.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net 1 Fluorescence Spectrophotometry

Radiative and Nonradiative Decay External conversion describes the process where the fluorophore loses electronic energy to its environment Pathways through collision with other solutes. Collisional quenching processes are particularly interesting as they allow the Radiative decay describes molecular deexcitation pro- biochemical environment of the fluorophores to be cesses accompanied by photon emission. Molecules in the measured. A number of important solute molecules, such excited electronic states can also relax by nonradiative as oxygen, are efficient fluorescence quenchers. Upon processes where excitation energy is not converted into collision, the fluorophore is deexcited nonradiatively. The photons but are dissipated by thermal processes such as collisional quenching rate can be expressed as: vibrational relaxation and collisional quenching. Let G and k be the radiative and nonradiative decay rates respectively kec 5 k0[Q] [7] and N be the fraction of fluorophore in the excited state. The temporal evolution of the excited state can be where k0 is related to the diffusivity and the hydrodynamics described by: radii of the reactants and [Q] is the concentration of the quencher. dN When collisional quenching is the dominant nonradia- À kN 1 dt tive process, eqn [1] predicts that fluorescence lifetime decreases with quencher concentration: ðÀþkÞt t=À N ¼ N0e ¼ N0e ½2 0 The fluorescence lifetime, t, of the fluorophore measures 1 k00Q 8 the combined rate of the radiative and nonradiative pathways: The steady state fluorescence intensity, F, also diminishes relative to the fluorescence intensity in the absence of 1 quencher, F0. This effect is described by the Stern–Volmer 3 À k equation: In the absence of nonradiative decay processes, one can F0 define the intrinsic lifetime of the fluorophore: 1 k00Q9 F 1 Fluorescence signal reduction can also result from ground 4 0 À state processes – steady state quenching. A fluorophore The ‘efficiency’ of the fluorophore can then be quantified can be chemically bound to a quencher to form a ‘dark by the fluorescence quantum yield, Q: complex’ – a product that does not fluoresce. Fluorescence intensity decreases with steady state quenching as: À Q 5 F0 À k 0 1 K Q10 F s where Ks is the association constant of the quencher and the fluorophore. Fluorescence lifetime is not affected by steady state quenching as the excited states are not Factors Affecting Fluorescence Intensity involved.

A number of factors contributes to the nonradiative decay pathways of the fluorophores and reduces fluorescence intensity. In general, the nonradiative decay processes can Phosphorescence be classified as: Intersystem crossing is another process where fluorescence signal is reduced and phosphorescence is generated. Spin- k 5 kic 1 kec 1 kis [6] orbit coupling is a quantum mechanical process that is where kic is the rate of internal conversion, kec is the rate of responsible for intersystem crossing. Intersystem crossing external conversion, and kis is the rate of intersystem describes the relaxation of the molecule from a singlet crossing. excited state to a lower energy, triplet excitation state. Internal conversion is a process where the electronic Since spin-orbit coupling is a weak effect, the intersystem energy is converted to the vibrational energy of the crossing rate is low. The relaxation from the triplet state to fluorophore itself. Since vibrational processes are driven the singlet ground state requires another change of by thermal processes, the internal conversion rate typically multiplicity. Hence, the decay from the triplet states also increases with temperature, which accounts for the has a very low rate. However, radiative relaxation, commonly observed decrease in fluorescence intensity with phosphorescence, does occur due to spin-orbit coupling. rising temperature. The typical phosphorescence lifetime is on the order of

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microseconds to seconds. Phosphorescence has larger EXO SC Stoke shift than fluorescence owing to the triple state having lower energy. LS Since phosphorescence rate is often much lower than thermally activated nonradiative decay processes such as collisional quenching, phosphorescence is rarely observed in aqueous systems at physiological temperature. How- ever, a number of protein conformation studies at cryogenic temperatures have utilized phosphorescence spectroscopy. EMO

Instrumentation for Fluorescence Spectrophotometry

The measurement of fluorescence signals provides a DET sensitive method of monitoring the biochemical environ- ment of a fluorophore. Instruments have been designed to Figure 2 A typical fluorometer design. LS is the light source, EXO is the measure fluorescence intensity, spectrum, lifetime and excitation optical train, SC is the specimen chamber, EMO is the emission polarization. optical train, and DET is the optical detector. Both the excitation and Fluorescence intensity measurement allows the determi- emission optical trains contain beam-shaping and collimation optics. For wavelength-resolved measurements, spectral selection optical nation of the presence of fluorophores and their concen- components such as monochrometers and filters are included in the EXO trations. Fluorescence intensity measurement is used in and EMO. For polarization measurement, polarizers are added to EXO and numerous biochemical assays. The instrument designs for EMO. For lifetime measurement, a laser light source is often used and high- these assays are rather straightforward but are as varied as speed electronics are integrated into the detector subsystem. the applications. Fluorometers are general-purpose instruments designed to measure fluorescence spectrum, polarization and/or pulsed lasers are often used as excitation light sources. A lifetime. A typical fluorometer includes a light source, a time-correlated single photon counting method is fre- specimen chamber with integrated optical components, quently employed, in which the time delay between the and high sensitivity detectors (Figure 2). The most common excitation light pulse and the resultant fluorescence photon light source for fluorometers are lamp sources, such as is measured by high-speed electronics. In the frequency xenon arc lamps. These lamps provide a relatively uniform domain, the specimen is excited by a light source with high- intensity over a broad spectral range from the ultraviolet to frequency content. The resultant fluorescence signal is also the near infrared. The optical paths of the excitation and modulated at the same frequency but is phase-delayed and the detection light paths are along the orthogonal axis. The amplitude-demodulated. The phase delay and demodula- orthogonal arrangement ensures minimal leakage of tion contain the lifetime information of the fluorophore excitation light into the detection side. High sensitivity and can be measured using heterodyning or homodyning photodetectors such as photomultipliers or charge- detection techniques. coupled device cameras are commonly used. For polarization measurement, polarizers are inserted For spectral measurement, monochrometers or band- into the excitation and emission light paths. With the pass filters are placed in the excitation and emission light excitation polarizer fixed, the emission polarizater can be paths to select a specific spectral band. The excitation rotated to measure the perpendicular (I\) and parallel (I6) spectrum is defined as the fluorescent intensity measured as components of the fluorescence emission. The steady state a function of excitation wavelength at a constant emission polarization is defined as: wavelength; the emission spectrum is the fluorescent intensity measured as a function of emission wavelength I I at a constant excitation wavelength. P 11 I I Fluorometers have also been designed to measure fluorescence lifetime. Given the typical lifetime of fluor- and an equivalent measure is the steady state anisotropy: ophores, accurate lifetime measurement requires photo- detectors and signal processing electronics with I I subnanosecond resolution. High-precision fluorometers r 12 I 2I have been designed in both the time and frequency domains. In the time domain, femtosecond or picosecond

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Applications of Fluorescence in the contains information related to the shape of the proteins. Diffusional restrictions of molecules in biological macro- Study of Biological Structure and structures, such as cellular membrane or the cytoskeleton, Function can also be quantified based on polarization measurement. The most common application of fluorescence polarization The use of fluorescence in biology and medicine is spectroscopy is the monitoring of protein–ligand binding ubiquitous. In particular, the measurements of fluores- and oligomerization. The combination of lifetime and cence spectrum, lifetime and polarization are powerful polarization measurements allows the quantification of methods of studying biological structure and function. rotational rate and has been used to study protein domain The fluorescence spectrum is highly sensitive to the motion. biochemical environment of the fluorophore. Fluoro- The limited scope of this article precludes a complete phores have been designed such that their spectra change discussion on the use of different spectroscopic methods to as a function of the concentration of metabolites, such as deduce biological structures and functions. However, as a pH and calcium. Fluorescence spectral changes resulting demonstration of the general principles, we will examine from solvent relaxation of fluorescent amino acids, such as the use of fluorescence polarization to monitor protein– tryptophan and tyrosin, are important reporters of protein ligand interactions. In a typical molecular binding assay, structure and folding. Protein domain structure and the smaller ligand molecules are labelled by a fluorophore. motion on the subnanometer scale can be spectrally The binding of the small ligand to a larger protein results in monitored using fluorescence resonance energy transfer a significant increase in the hydrodynamic radius of the (FRET). FRET is a nonradiative process where the energy composite particle and a slower rotational diffusion rate. is transferred between two fluorophores. FRET requires The change in rotational diffusion rate can be measured that the emission spectrum of one fluorophore (the donor) using fluorescence polarization assay. The fraction of overlaps the absorption spectrum of a second fluorophore bound molecules can be estimated by quantifying the (the acceptor). The efficiency of this process is a strong optical signal contributions from the fast and slow function (1/r6) of the molecules’ relative distance, r. Protein diffusers. The association constant of this protein–ligand conformation can be monitored by labelling the relevant interaction can also be measured by quantifying the structures with a FRET pair. The distance between the two fractions of bound and free proteins at different protein– fluorophores can be quantified by spectrally resolving the ligand mixing ratios. relative fluorescence intensities of the donor and the Fluorescence spectophotometry is widely used in many acceptor. areas of biology and medicine. A basic understanding of Fluorescence lifetime provides complementary informa- fluorescence principles, fluorophores properties, instru- tion to spectral measurement. Many fluorophores may ments and techniques is a prerequisite to the study of a wide respond to environmental changes with lifetime variations. range of biological systems. An important example is oxygen concentration measure- ment based on the dynamic quenching of long-lifetime fluorophores. Lifetime measurements are also used to distinguish dynamic and static quenching mechanisms. Further Reading Lifetime-resolved FRET measurement allows the determi- Becker RS (1969) Theory and Interpretation ofFluorescence and nation of distance distribution of a population of FRET Phosphorescence. New York: Wiley. pairs. Birks JB (1970) Photophysics ofAromatic Molecules . New York: Wiley. Fluorescence polarization measures the rotational Lakowicz JR (1999) Principles ofFluorescence Spectroscopy . New York: diffusion rate of macromolecules. Rotational diffusion Plenum Press.

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