Fluorescence spectroscopy

Fluorescence spectroscopy or fluorometry or spectrofluorimetry is a type of electromagnetic spectroscopy which analyzes fluorescence 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 spectroscopy.

Devices that measure fluorescence are called fluorometers or fluorimeters.

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Theory

Main article: Fluorescence

Molecules have various states referred to as energy levels. Fluorescence spectroscopy is primarily concerned with electronic states and vibrational states. Generally, the species being examined will have a ground electronic state (a low energy state) of interest, and an excited electronic state of higher energy. Within each of these electronic states are various vibrational states.

Photons are small "packets" of radiation, each with an energy proportional to its frequency; photons of high frequency light have higher energy than those of low frequency light. These can be absorbed by molecules, with the molecule gaining the energy of the photon, or emitted by molecules, with the photon carrying some of the energy of the molecule away.

In fluorescence spectroscopy, the species is first excited, by absorbing a photon of light, 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 frequencies of fluorescent light emitted by a sample are measured, holding the excitation light at a constant wavelength. This is called an emission spectrum. An excitation spectrum is measured by recording a number of emission spectra using different wavelengths of excitation light.

Instrumentation

Two general types of instruments exist:

• Filter fluorometers use filters to isolate the incident light and fluorescent light.
• Spectrofluorometers use diffraction grating monochromators to isolate the incident light and fluorescent light.

Both types utilize 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 arcs and mercury vapor lamps 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 diffraction grating, that is, collimated light enters a grating and exits with a different angle depending on the wavelength. The monochromator can then 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 light, 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 ^[1], when compared to the 180° geometry. Furthermore, the fluorescence can also be measured from the front, which is often done for turbid 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. ^[2]

Analysis of data

At low concentrations the fluorescence intensity will generally be proportional to the concentration of the fluorophore.

Unlike in UV/visible spectroscopy, ‘standard’, device independent spectra are not easily attained, however. 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 frequencies. 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.

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 state, the molecules may relax back to a vibrational level other than the vibrational ground state ^[3]. 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. ^[4][5]

Tryptophan Fluorescence

Tryptophan 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, surfactants 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 aqeuous 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 aqeuous solvent will cause a blue shifted emission spectrum if the tryptophan is embedded in the surfactant vesicle or micelle ^[6].

At 295 nm, the tryptophan emission spectrum is dominant over the weaker tyrosine and phenylalanine fluorecence.

Applications

Fluorescence spectrocopy is used in, among others, biochemical, medical, and chemical research fields for analyzing organic compounds. There has also been a report of its use in differentiating malign skin tumors from benign.

References

1. ^ Rendell, D. (1987). Fluorescence and Phosphorescence. Crown
2. ^ Sharma, A. and Schulman, S. G. (1999). Introduction to Fluorescence Spectroscopy. Wiley interscience.
3. ^ Gauglitz, G. and Vo-Dinh, T. (2003). Handbook of spectroscopy. Wiley-VCH.
4. ^ Lakowicz, J. R. (1999). Principles of Fluorescence Spectroscopy. Kluwer Academic / Plenum Publishers
5. ^ Sharma, A. and Schulman, S. G. (1999). Introduction to Fluorescence Spectroscopy. Wiley interscience.
6. ^ Caputo GA, London E. Cumulative effects of amino acid substitutions and hydrophobic mismatch upon the transmembrane stability and conformation of hydrophobic alpha-helices. Biochemistry. 2003 Mar 25;42(11):3275-85.

See also

• Confocal microscopy
• Fluorescence correlation spectroscopy
• X-ray fluorescence spectroscopy
• Beer's law
• Dye tracing