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
. Instrumentation for Fluorescence Spectrophotometry
Fluorescence spectrophotometry is a class of techniques that assay the state of a biological
. 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.

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 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.
 When collisional quenching is the dominant nonradia-

   3
 tive process, eqn [1] predicts that fluorescence lifetime
decreases with quencher concentration:
N ¼ N0 eð þkÞt ¼ N0 et= ½2
6
The fluorescence lifetime, t, of the fluorophore measures
3  6 6 
 B
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
3 quencher, F0. This effect is described by the Stern–Volmer

5 equation:

In the absence of nonradiative decay processes, one can 6
define the intrinsic lifetime of the fluorophore:
3  6 6 
 D

3 Fluorescence signal reduction can also result from ground
6
7 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:




; 6
 6
3   
 36

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
k 5 kic 1 kec 1 kis [6] signal is reduced and phosphorescence is generated. Spin-
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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 Fluorescence Spectrophotometry

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
emission optical trains contain beam-shaping and collimation optics. For
Fluorescence intensity measurement allows the determi-
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   
at a constant excitation wavelength. 
33
  
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   
subnanosecond resolution. High-precision fluorometers 
3I
  I
have been designed in both the time and frequency
domains. In the time domain, femtosecond or picosecond

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 Fluorescence Spectrophotometry

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 of Fluorescence and
nation of distance distribution of a population of FRET Phosphorescence. New York: Wiley.
pairs. Birks JB (1970) Photophysics of Aromatic Molecules. New York: Wiley.
Fluorescence polarization measures the rotational Lakowicz JR (1999) Principles of Fluorescence Spectroscopy. New York:
diffusion rate of macromolecules. Rotational diffusion Plenum Press.

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