Fluorescence spectroscopy is routinely used for studying structural changes in conjugated systems, aromatic molecules, and rigid, planar compounds due to alterations in temperature, pH, ionic strength, solvent, and ligands. A single fluorophore can generate thousands of detectable photons that can be repeatedly excited and detected, making fluorescence spectroscopy is a highly sensitive technique.
Fluorescence is a type of radiative emission that occurs when a molecule absorbs energy at a wavelength where it has a transition dipole moment. The excitation energy provided to the molecule at the ground state promotes photons to an excited singlet state, where they then decay to the lowest vibrational energy level of this excited singlet state. This energy further relaxes back to the ground state of the molecule, emitting photons in the process, as shown in Figure 1.
Fluorescent molecules can also undergo there are three methods of nonradiative relaxation where the excitation energy is not converted into photons: (1) internal conversion, (2) external conversion, and (3) intersystem crossing. Internal conversion occurs when there is a relatively small energy gap between two electronic states and the electrons transition from a higher electronic state to one of lower energy. Here the energy is transferred to the vibrational modes of the electronic state. Since vibrational processes are thermally driven, increasing temperature leads to decreases in fluorescence intensity. In external conversion, energy is lost through collisional quenching with solute molecules in the fluorophore’s environment. Intersystem crossing arises when vibrational levels of the singlet and triplet excited states overlap in energy and electrons transition from the lowest singlet excited state to the first excited triplet state. The photons emitted as they return back to the ground state is known as phosphorescence (Figure 1). The triplet state is lower in energy than the singlet state so phosphorescence peaks are found at longer wavelengths than fluorescence. Since these transitions are also forbidden, phosphorescence exhibits a longer lifetime (~10-4 – 102 seconds) compared with fluorescence (~10-9 – 10-6 seconds). The longer lifetimes also lead to thermal deactivation via oxygen quenching, solvent movement, and intermolecular collision so phosphorescence typically cannot be observed at room temperature and samples must therefore be cooled at liquid nitrogen temperature.
Beer’s Law and Concentration Effects
While absorption occurs on the timescale of less than 10-15 seconds, the relaxation process from the excited to the ground state is much slower. Therefore, fluorescence can provide information on a fluorophores’ interactions with surrounding molecules and solvents, unlike absorption.
Fluorescence intensity is directly proportional to the excitation light intensity
F=2.303 * K * I0 * εbc
where K is a constant based on instrument geometry, I0 is the intensity of the excitation light, e is the fluorophore’s molar absorptivity, b is the pathlength, and c is the concentration. Since the fluorescence intensity is not ratioed to the incident light intensity like with absorption measurements, the fluorescence sensitivity is much greater because it is not limited by the instruments ability to differentiate between the incident and detected intensities. Consequently, smaller concentrations are required for measurements.
The above equation is only linear when the sample absorbance is less than 0.05 AU. If a sample is too concentrated, the emission light can be reabsorbed by the fluorophore, attenuating the fluorescence signal at shorter wavelengths. Excitation light may also not fully penetrate the full width of a highly concentrated sample, which will also lead to decreased fluorescence intensities.
INSTRUMENTATION OF FLUORESCENCE SPECTROSCOPY
Characteristics of a Fluorescence Spectrum
Fluorometers are composed of an excitation and emission monochromator, allowing users to obtain both excitation and emission spectra. A measurement made by a fluorometer is unique to the individual instrument’s excitation and emission monochromators. Fluorescence is directly related to luminous flux and the efficiency of measurement and therefore dependent on the instrument design and components such as the light source, monochromator optics, and photomultiplier tube. Each light source will have a different spectral output (both shape and power) which will vary and decrease over the lifetime of the source.
Excitation spectra plot the intensity at a fixed emission wavelength while varying the excitation wavelengths. Since most emission spectra are independent of the excitation wavelength, the excitation spectra are frequently duplicates of the fluorophore’s absorption spectrum.
Conversely, an emission spectrum plot the intensity at a fixed excitation wavelength while scanning through varying emission wavelengths. These emission scans provide information on the molecular structure of the fluorophore and the local environment surrounding it. Since the fluorescence emission always occurs from the lowest excited state to the ground state, the shape of the emission spectrum is independent of the excitation wavelength. More energy is also required to excite a molecule from the ground to the excited state, resulting in emission peaks at longer wavelengths (ie smaller energies) than their corresponding excitation wavelengths. This difference in energy between the excitation and emission wavelengths is known as the Stokes shift.
In addition, absorption and emission spectra are frequently mirror images of one another due to the equal distribution between the vibrational energy levels of the excited and ground states (Figure 3). The Franck-Condon principle explains that because the nuclei are relatively large and the electronic transition involved in emission and absorption occur on such fast timescales, there is no time for nuclei to move and the vibrational energy levels and therefore remain roughly the same throughout the electronic transition.
Since the fluorescence intensity is proportional to the input light intensity, the amount of light passed through the monochromator will greatly affect the intensity. The sum of the excitation and emission bandwidths should be about the spectral bandwidth (SBW) of the peak being monitored so that all peaks are well resolved. As long as this rule of thumb is followed, the bandwidths can be opened to increase the amount of light throughput for samples with low fluorescence. The SBW can also be impacted by the Stokes shift of the fluorophore. Narrower Stokes shifts may limit the range of acceptable SBWs that can be used.
Scattered light can give rise to artifacts, distorting the fluorescence spectrum. The three most common types of scatter seen in fluorescence are Rayleigh, 2nd order, and Raman scatter (Figure 3). Rayleigh scattering is the scattered excitation light and therefore peaks at the excitation wavelength. 2nd order scatter is higher-order scatter observed at twice the excitation wavelength. Raman scattering is inelastic scatter due to solvents and peaks at a fixed energy from the excitation wavelength. To differentiate Raman scattering from a fluorescence peak, the excitation wavelength can be varied in 5 to 10 nm increments and if the peak in question shifts with the excitation wavelength and decreases in intensity, then that peak, is due to Raman scatter. You can also check to see if the peak is in the blank solvent spectrum. If it is, there is a chance it is a Raman peak. If the fluorescence peak is too close or overlapping with either the Raman or Rayleigh scatter, the bandwidths and/or excitation wavelength can be adjusted to shift the scatter off the fluorescence peak. These effects are most prominent for very low fluorophore concentrations and especially highly scattering solutions, like proteins, microsphere, nanoparticles, as well as solids.
The Automatic Gain Control function automatically adjusts the gain of a signal from the detector based on the fluorescence intensity. This optimizes the signal to noise throughout the entire scanned range for spectral or time course measurements so that peaks with different intensities are automatically adjusted to improve the S/N and assure result accuracy.
Automatic Sensitivity Control System (SCS) expands the dynamic range of the detected fluorescence signal by automatically adjusting the detector voltage according to the fluorescence intensity. This allows for fixed wavelength or quantitative analyses measurements of sub-picomolar to micromolar concentrations without manually changing the instrument.
Figure 5. Calibration curve of fluorescein solutions from 5·10-13 to 1.5·10-6 M using the auto-SCS function.
APPLICATIONS OF FLUORESCENCE SPECTROSCOPY
Fluorescence anisotropy is observed when a fluorophore emits light of different intensities depending on the axes of polarization and is described by the following equation
where is the emission intensity parallel to the excitation plane and is the emission intensity perpendicular to the excitation plane. G is called the G-factor or instrument grating factor and accounts for the polarization dependence of the emission monochromator.
All fluorophores have transition moments that occur along specific directions along the molecular axis. When exposed to polarized light, the randomly oriented fluorophores that have their absorption transition moments oriented around the angle of the incident light will be excited and this excited state population is partially oriented. As a molecule returns from an excited state back to its ground state, the electron charge is redistributed and the change in the orientation of the dipole moments effect the excitation and emission polarizations. For example, when fluorescence is emitted before a molecule rotates, the fluorescence light will be strongly polarized towards the direction of the excitation light’s polarization. If the light is emitted after the rotation of the molecule in a completely random direction, the fluorescence will be no longer polarized.
When measuring fluorescence anisotropy, the following factors will affect the molecular movement: (1) molecular size, (2) viscosity of the molecule’s environment, and (3) strength and degrees of freedom of a bound molecule. Anisotropy measurements determine the average angular displacement of a fluorophore that occurs between the absorption and emission of a photon. The angular displacement is dependent on the rate and extent of the rotational diffusion during the lifetime of the excited state. When a fluorophore is unrestricted and allowed to freely rotate before re-emitting a photon, the rate of diffusion is generally faster than the rate of the emission and the anisotropy is roughly equal to zero. Rotational diffusion changes of the direction of the transition moment which depolarizes the emission. The more restricted the fluorophore, the larger the anisotropy value will be since the decrease in flexibility will decrease the overall rate of rotation.
Fluorescence resonance energy transfer (FRET) is a mechanism governing the energy transfer between two neighboring molecules. A donor, initially in its excited state, may transfer energy to an acceptor molecule through non-radiative electron resonance.
FRET is monitored by the spectrofluorometer, which measures the fluorescence/quenching of acceptor or excited donor. FRET efficiency depends on the following factors: the distance between the donor and acceptor, the spectral overlap between the donor and acceptor, and the alignment of their dipole moments. The efficiency is inversely proportional to the sixth power of the distance between the donor and acceptor, making the technique extremely sensitive to small changes in distance. When the overlap area of the donor fluorescence spectrum and the acceptor absorption spectrum is larger, FRET efficiency is higher. The FRET efficiency is also at a maximum when the two dipole moments are parallel or anti-parallel to each other, and no energy transfer occurs when the dipole moments are perpendicular to one another. Typically when the distance between the donor and acceptor is betwen1 and 10 nm, FRET occurs.
Quantum Yield and Spectral Correction
Different molecular and environmental conditions not only effect whether a molecule will fluoresce or not, but can also determine the intensity or quantum yield of the emitted fluorescence radiation. A molecule’s efficiency to fluoresce is described by its quantum yield and is defined as the ratio of the number of photons absorbed to the number of photons emitted by the sample.
In some cases it is necessary to determine accurate spectral measurement. This is made using references to known calibrated materials. The calibrated sources that are used are checked for absolute spectral output on a known instrument and a reference spectrum is supplied to correct the individual instrument supplied to the customer. For Spectral Correction to work effectively, it has to be performed at each instrument parameter and bandwidth combinations so the Spectral Correction at a 5 nm spectral bandwidth cannot be applied to measurement using a 10 nm SBW. This applies to the position of polarizers if they are being used, as well as the use of higher order filters. It is necessary to perform the Spectral Correction for each combination of spectral bandwidths to be used by the customer, for the inclusion or exclusion of the higher order filter selection, and for the positions of polarizers if fitted. The sample excitation and emission wavelengths will determine what solution/light source is used for calibration.
For applications probing the NIR region of the spectrum, the spectral response of the PMT detector is critical for obtaining data. In the red end of the visible region, into the NIR, the quantum efficiency of the PMT significantly decreases, resulting in little to no signal intensity during sample measurements. FRET experiments and NIR dyes and probes are frequently monitored at wavelengths above 500 nm and in many cases have small signals, even for such a sensitive technique like fluorescence. Figure 8 illustrates the difference in fluorescence intensity of rhodamine B using a standard PMT compared with a PMT that is more sensitive to wavelengths on the red end of the spectrum.