Fluorescence spectroscopy aka fluorometry or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes
fluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light of a lower energy, typically, but not necessarily, visible light. A complementary technique is
absorption spectroscopyAbsorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of the absorption varies as a...
.
Devices that measure
fluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
are called
fluorometerA fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium...
s or fluorimeters.
Theory
Molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states. Generally, the species being examined has a
ground electronic stateIn quantum mechanics, a stationary state is an eigenvector of the Hamiltonian, implying the probability density associated with the wavefunction is independent of time . This corresponds to a quantum state with a single definite energy...
(a low energy state) of interest, and an excited electronic state of higher energy. Within each of these electronic states are various vibrational states.
In fluorescence spectroscopy, the species is first excited, by absorbing a
photonIn physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...
, from its ground electronic state to one of the various vibrational states in the excited electronic state. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state of the excited electronic state.
The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As molecules may drop down into any of several vibrational levels in the ground state, the emitted photons will have different energies, and thus frequencies. Therefore, by analysing the different frequencies of light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined.
In a typical experiment, the different wavelengths of fluorescent light emitted by a sample are measured using a
monochromatorA monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input...
, holding the excitation light at a constant wavelength. This is called an
emission spectrum. An
excitation spectrum is the opposite, whereby the emission light is held at a constant wavelength, and the excitation light is scanned through many different wavelengths (via a monochromator). An
emission map is measured by recording the emission spectra resulting from a range of excitation wavelengths and combining them all together. This is a three dimensional surface data set: emission intensity as a function of excitation and emission wavelengths, and is typically depicted as a contour map.
Instrumentation
Two general types of instruments exist:
- Filter fluorometer
A filter fluorometer is a type of fluorometer that may be employed in fluorescence spectroscopy.In the fluorometer, a light source emits light of an excitation wavelength that is relevant to the compound to be measured. Filter fluorometers produce specific excitation and emission wavelengths by...
s use filters to isolate the incident light and fluorescentFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
light.
- Spectrofluorometer
The spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample...
s use diffraction gratingIn optics, a diffraction grating is an optical component with a periodic structure, which splits and diffracts light into several beams travelling in different directions. The directions of these beams depend on the spacing of the grating and the wavelength of the light so that the grating acts as...
monochromatorA monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input...
s to isolate the incident light and fluorescent light.
Both types use the following scheme: The light from an excitation source passes through a filter or monochromator, and strikes the sample. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescent light is emitted in all directions. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90° to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector.
Various light sources may be used as excitation sources, including lasers, photodiodes, and lamps;
xenon arcsA xenon arc lamp is a specialized type of gas discharge lamp, an electric light that produces light by passing electricity through ionized xenon gas at high pressure to produce a bright white light that closely mimics natural sunlight...
and
mercury-vapor lampA mercury-vapor lamp is a gas discharge lamp that uses an electric arc through vaporized mercury to produce light. The arc discharge is generally confined to a small fused quartz arc tube mounted within a larger borosilicate glass bulb...
s in particular. A laser only emits light of high irradiance at a very narrow wavelength interval, typically under 0.01 nm, which makes an excitation monochromator or filter unnecessary. The disadvantage of this method is that the wavelength of a laser cannot be changed by much. A mercury vapor lamp is a line lamp, meaning it emits light near peak wavelengths. By contrast, a xenon arc has a continuous emission spectrum with nearly constant intensity in the range from 300-800 nm and a sufficient irradiance for measurements down to just above 200 nm.
Filters and/or monochromators may be used in fluorimeters. A monochromator transmits light of an adjustable wavelength with an adjustable tolerance. The most common type of monochromator utilizes a diffraction grating, that is, collimated light illuminates a grating and exits with a different angle depending on the wavelength. The monochromator can then be adjusted to select which wavelengths to transmit. For allowing anisotropy measurements the addition of two polarization filters are necessary: One after the excitation monochromator or filter, and one before the emission monochromator or filter.
As mentioned before, the fluorescence is most often measured at a 90° angle relative to the excitation light. This geometry is used instead of placing the sensor at the line of the excitation light at a 180° angle in order to avoid interference of the transmitted excitation light. No monochromator is perfect and it will transmit some
stray lightStray light is light in an optical system, which was not intended in the design. The light may be from the intended source, but follow paths other than intended, or it may be from a source other than the intended source...
, that is, light with other wavelengths than the targeted. An ideal monochromator would only transmit light in the specified range and have a high wavelength-independent transmission. When measuring at a 90 angle, only the light scattered by the sample causes stray light. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000, when compared to the 180° geometry. Furthermore, the fluorescence can also be measured from the front, which is often done for turbid or opaque samples
.
The detector can either be single-channeled or multichanneled. The single-channeled detector can only detect the intensity of one wavelength at a time, while the multichanneled detects the intensity at all wavelengths simultaneously, making the emission monochromator or filter unnecessary. The different types of detectors have both advantages and disadvantages.
The most versatile fluorimeters with dual monochromators and a continuous excitation light source can record both an excitation spectrum and a fluorescence spectrum. When measuring fluorescence spectra, the wavelength of the excitation light is kept constant, preferably at a wavelength of high absorption, and the emission monochromator scans the spectrum. For measuring excitation spectra, the wavelength passing though the emission filter or monochromator is kept constant and the excitation monochromator is scanning. The excitation spectrum generally is identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption.
Analysis of data
At low concentrations the
fluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
intensityIn physics, intensity is a measure of the energy flux, averaged over the period of the wave. The word "intensity" here is not synonymous with "strength", "amplitude", or "level", as it sometimes is in colloquial speech...
will generally be proportional to the
concentrationIn chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
of the
fluorophoreA fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
.
Unlike in UV/visible spectroscopy, ‘standard’, device independent spectra are not easily attained. Several factors influence and distort the spectra, and corrections are necessary to attain ‘true’, i.e. machine-independent, spectra. The different types of distortions will here be classified as being either instrument- or sample-related. Firstly, the distortion arising from the instrument is discussed. As a start, the light source intensity and wavelength characteristics varies over time during each experiment and between each experiment. Furthermore, no lamp has a constant intensity at all wavelengths. To correct this, a beam splitter can be applied after the excitation monochromator or filter to direct a portion of the light to a reference detector.
Additionally, the transmission efficiency of monochromators and filters must be taken into account. These may also change over time. The transmission efficiency of the monochromator also varies depending on wavelength. This is the reason that an optional reference detector should be placed after the excitation monochromator or filter. The percentage of the fluorescence picked up by the detector is also dependent upon the system. Furthermore, the detector quantum efficiency, that is, the percentage of photons detected, varies between different detectors, with wavelength and with time, as the detector inevitably deteriorates.
Two other topics that must be considered include the optics used to direct the radiation and the means of holding or containing the sample material (called a cuvette or cell). For most UV, visible, and NIR measurements the use of precision quartz cuvettes is necessary. In both cases, it is important to select materials that have relatively little absorption in the wavelength range of interest. Quartz is ideal because it transmits from 200 nm-2500 nm; higher grade quartz can even transmit up to 3500 nm, whereas the absorption properties of other materials can mask the fluorescence from the sample.
Correction of all these instrumental factors for getting a ‘standard’ spectrum is a tedious process, which is only applied in practice when it is strictly necessary. This is the case when measuring the quantum yield or when finding the wavelength with the highest emission intensity for instance.
As mentioned earlier, distortions arise from the sample as well. Therefore some aspects of the sample must be taken into account too. Firstly, photodecomposition may decrease the intensity of fluorescence over time. Scattering of light must also be taken into account. The most significant types of scattering in this context are Rayleigh and Raman scattering. Light scattered by Rayleigh scattering has the same wavelength as the incident light, whereas in Raman scattering the scattered light changes wavelength usually to longer wavelengths. Raman scattering is the result of a virtual electronic state induced by the excitation light. From this
virtual stateThe term virtual state is commonly used to refer to two different types of states in physical systems. It may refer to a very short-lived, unobservable quantum state or a real, but unstable, state...
, the molecules may relax back to a vibrational level other than the vibrational ground state. In fluorescence spectra, it is always seen at a constant wavenumber difference relative to the excitation wavenumber e.g. the peak appears at a wavenumber 3600 cm
−1 lower than the excitation light in water.
Other aspects to consider are the inner filter effects. These include reabsorption. Reabsorption happens because another molecule or part of a macromolecule absorbs at the wavelengths at which the fluorophore emits radiation. If this is the case, some or all of the photons emitted by the fluorophore may be absorbed again. Another inner filter effect occurs because of high concentrations of absorbing molecules, including the fluorophore. The result is that the intensity of the excitation light is not constant throughout the solution. Resultingly, only a small percentage of the excitation light reaches the fluorophores that are visible for the detection system. The inner filter effects change the spectrum and intensity of the emitted light and they must therefore be considered when analysing the emission spectrum of fluorescent light.
Tryptophan fluorescence
The
fluorescenceFluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
of a folded protein is a mixture of the fluorescence from individual aromatic residues. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of
tryptophanTryptophan is one of the 20 standard amino acids, as well as an essential amino acid in the human diet. It is encoded in the standard genetic code as the codon UGG...
residues, with some emissions due to tyrosine and phenylalanine; but disulfide bonds also have appreciable absorption in this wavelength range. Typically, tryptophan has a wavelength of maximum absorption of 280 nm and an emission peak that is solvatochromic, ranging from ca. 300 to 350 nm depending in the polarity of the local environment Hence, protein fluorescence may be used as a diagnostic of the conformational state of a protein. Furthermore, tryptophan fluorescence is strongly influenced by the proximity of other residues (
i.e., nearby
protonated groups such as Asp or Glu can cause
quenchingQuenching refers to any process which decreases the fluorescence intensity of a given substance. A variety of processes can result in quenching, such as excited state reactions, energy transfer, complex-formation and collisional quenching. As a consequence, quenching is often heavily dependent on...
of Trp fluorescence). Also, energy transfer between tryptophan and the other fluorescent amino acids is possible, which would affect the analysis, especially in cases where the Förster acidic approach is taken. In addition, tryptophan is a relatively rare amino acid; many proteins contain only one or a few tryptophan residues. Therefore, tryptophan fluorescence can be a very sensitive measurement of the conformational state of individual tryptophan residues. The advantage compared to extrinsic probes is that the protein itself is not changed. The use of intrinsic fluorescence for the study of protein conformation is in practice limited to cases with few (or perhaps only one) tryptophan residues, since each experiences a different local environment, which gives rise to different emission spectra.
TryptophanTryptophan is one of the 20 standard amino acids, as well as an essential amino acid in the human diet. It is encoded in the standard genetic code as the codon UGG...
is an important intrinsic fluorescent probe (amino acid), which can be used to estimate the nature of microenvironment of the tryptophan. When performing experiments with denaturants,
surfactantSurfactants are compounds that lower the surface tension of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid...
s or other amphiphilic molecules, the microenvironment of the tryptophan might change. For example, if a protein containing a single tryptophan in its 'hydrophobic' core is denatured with increasing temperature, a red-shift emission spectrum will appear. This is due to the exposure of the tryptophan to an aqueous environment as opposed to a hydrophobic protein interior. In contrast, the addition of a surfactant to a protein which contains a tryptophan which is exposed to the aqueous solvent will cause a blue shifted emission spectrum if the tryptophan is embedded in the surfactant
vesicleA vesicle is a bubble of liquid within another liquid, a supramolecular assembly made up of many different molecules. More technically, a vesicle is a small membrane-enclosed sack that can store or transport substances. Vesicles can form naturally because of the properties of lipid membranes , or...
or
micelleA micelle is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions in contact with surrounding solvent, sequestering the hydrophobic single tail regions in the micelle centre. This phase is...
. Proteins that lack tryptophan may be coupled to a
fluorophoreA fluorophore, in analogy to a chromophore, is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength...
.
At 295 nm, the tryptophan emission spectrum is dominant over the weaker
tyrosineTyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
and
phenylalaninePhenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
fluorescence.
Applications
Fluorescence spectrocopy is used in, among others, biochemical, medical, and chemical research fields for analyzing
organic compoundAn organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
s. There has also been a report of its use in differentiating malignant, bashful skin tumors from benign.
Atomic Fluorescence Spectroscopy (AFS) techniques are useful in other kinds of analysis/measurement of a compound present in air or water, or other media, such as CVAFS which is used for heavy metals detection, such as mercury.
Fluorescence can also be used to redirect photons, see fluorescent solar collector.
External links