Luminescence spectroscopy deals with emission associated with a transition from an excited electronic state to a lower state (generally the ground state) (Lakowicz, 1999; Lakowicz, 1991-2000). Biological molecules at room temperature exhibit fluorescence. Phosphorescence from a triplet excited state to the singlet ground state is rarely observed at room temperature. One-photon absorption produces a fluorescence band that is red-shifted (to a lower energy). This shift between the peak of the absorption band and that of the fluorescence band is called Stokes shift. The amount of Stokes shift is a measure of the relaxation process occurring in the excited state, populated by absorption. The difference in the energy of the absorbed photon and that of the emitted photon corresponds to the energy loss due to nonradiative processes. The Stokes shift may arise from environmental effect as well as from a change in the geometry of the emitting excited state. Figure 4.9 shows the absorption and the emission spectra of fluorescein, a commonly used dye.
Although fluorescence measurements are more sophisticated than an absorption (transmission) experiment, they provide a wealth of the information about the structure, interaction, and dynamics in a bioassembly. Also, fluorescence imaging is the dominant optical bioimaging technique for biophotonics.
Figure 4.9. Absorption and fluorescence spectra of fluorescein in buffer (pH 9.0).
Figure 4.9. Absorption and fluorescence spectra of fluorescein in buffer (pH 9.0).
The fluorescence spectroscopy includes the study of the following features to probe the interaction and dynamics:
• Fluorescence spectra
• Fluorescence excitation spectra
• Fluorescence lifetime
• Fluorescence quantum efficiency
• Fluorescence depolarization
The fluorescence spectrum is obtained by exciting the molecules in a medium using a conventional lamp (a xenon lamp or a mercury xenon lamp). For excitation, a wavelength range corresponding to the absorption band is selected by a broad-band cutoff filter that only allows light at frequencies higher than that of emission. The fluorescence spectrum comprised of the fluorescence intensity as a function of frequency is obtained in a fluorescence spectrometer which includes a dispersive element (grating). Lasers are often used as a convenient and powerful source for one-photon excited fluorescence in which case it is called laser-induced fluorescence (LIF).
The fluorescence excitation spectra (sometimes simply referred to as excitation spectra) give information on the absorption (excitation) to the state that produces maximum fluorescence. Here the total fluorescence or fluorescence at the maximum frequency is monitored and the excitation frequency of a lamp or a tunable laser source is scanned to obtain the excitation spectrum. A maximum in the excitation spectrum corresponds to the frequency of a photon, where absorption produces maximum fluorescence.
Fluorescence lifetime represents the decay of fluorescence intensity. A simple fluorescence decay is exponential (first-order kinetics) involving a rate constant k which describes the decay of the fluorescence intensity I as I = I0e~kt where Io is the fluorescence intensity at the start of fluorescence (at t = 0). This behavior is called a single exponential decay. The rate constant k has two contributions, a radiative decay constant kr characterized by a radiative lifetime tr and a nonradiative decay constant knr, characterized by a nonra-diative lifetime tnr. Thus:
From experimental measurements and fit of the decay to a single exponential, one obtains the overall fluorescence lifetime t.
The radiative lifetime tr is inversely proportional to the strength of the transition dipole moment. It can be shown that it is related to the maximum extinction coefficient, emax(v), of the absorption to the emitting state as follows:
In this equation, emax(v) is in the unit of Lmol-1cm-1.
Two methods of measurement of fluorescence lifetimes are:
(a) Time Domain Measurement. Here a short pulse, generally from a pulse laser source, excites the fluorescence, and decay of fluorescence is measured. The fluorescence lifetimes are generally in the range of nanoseconds to hundreds of picoseconds. For nanosecond decay, one utilizes a fast scope or a boxcar technique, whereas for lifetimes in hundreds of picoseconds, one utilizes a streak camera.
(b) Phase Modulation Measurement. This method utilizes a modulated excitation source (a lamp or a mode-locked laser, the latter of which is discussed in Chapter 5) and is based on the principle that a finite fluorescence lifetime causes the fluorescence waveform to be phase-shifted by an amount j with respect to the waveform of the exciting light. This phase shift j is related to the lifetime by the following equation:
where w is the modulation frequency (rate of modulation of exciting light). Therefore, from a measurement of the phase shift using a phasesensitive detector (a lock-in amplifier), one can obtain the fluorescence lifetime t. Several companies now sell instruments for phase-modulation lifetime measurements.
A rapidly growing field in photobiology is time-resolved fluorescence spectroscopy. Here the entire fluorescence spectrum is obtained as a function of time to monitor a spectral change induced by any dynamic change in the local configuration of the fluorescent unit called fluorophore or fluorochrome.
A nonexponential decay or a multiexponential decay (fit into a weighted sum of a number of exponentials) represents more complicated decay kinetics of the excited states. Some of the processes are (i) decay of the excited states through a number of channels (to different lower states), (ii) bimolec-ular decay involving interaction between two molecules, (iii) diffusion-controlled decay, and (iv) Förster energy transfer from an excitation donor molecular unit (the molecule absorbing the photon) to an excitation acceptor (the molecule which accepts the excitation and then may emit). Förster energy transfer is efficient when the emission spectrum of the donor molecule overlaps with the absorption spectrum of the acceptor molecule. With a significant overlap, the energy transfer is also called a resonance energy transfer and the fluorescence from the acceptor molecule is also called fluorescence resonance energy transfer (FRET). FRET has also found useful application for bioimaging, as discussed in Chapters 7 and 8 on bioimaging.
The rate of energy-transfer under a dipole-dipole transfer mechanism is inversely proportional to the sixth power of their separation. This dependence of energy transfer has been used to determine distance of separation between the excitation donor and acceptor sites and their mobilities.
The fluorescence quantum efficiency (also called quantum yield) O is defined as
The quantum yield is a quantitative measure of the ratio of the number of photons emitted to the number of photons absorbed. In the absence of any nonradiative decay, the quantum yield O equals 1; that is, the excited state decays only by a radiative (fluorescence) process. This is the case producing the most efficient fluorescence; therefore, ideal fluorophores to be used as fluorescent probes should have a quantum yield as close as possible to 1. Fluorescence efficiency (quantum yield) serves as an excellent probe for the environment surrounding a fluorophore in a bioassembly.
Fluorescence depolarization is a measure of the loss of polarization of fluorescence by a number of dynamic effects such as rotation of the fluorophore.
The polarization P of fluorescence is defined as
Another quantity also representing polarization of fluorescence is called fluorescence emission anisotropy, defined as r - </N - /1)/(/n + 2IJ. Here I and I1
are the fluorescence intensities polarized parallel and perpendicular to the polarization of excitation light.
The polarization ratio is determined by the relative orientation of the transition dipole moment (a vector) connecting the emitting excited state to the ground state (also called emission dipole) and the transition dipole moment connecting the ground state to the absorbing excited state (also called absorption dipole). For a randomly oriented rigid medium (molecular not being able to change the orientation) averaging over all possible molecular orientation yields P = +1/2 for the case when absorption (excitation) and emission dipoles are parallel, and P = -1/3 for the case when they are perpendicular to each other. A significant reduction in the magnitude of P indicates fluorescence depolarization. Therefore, a study of P or r for a fluorophore attached to a biopolymer or a biomembrane can provide information about the rotational mobility of its microenvironment. The P and r measurements are also used to measure rotational diffusion of molecules in biological systems such as membranes and cytosols (the biological systems are described in Chapter 3).
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