Fluorescence spectroscopy measures the intensity of photons emitted from a sample after it has absorbed photons. Most fluorescent molecules are aromatic. Fluorescence is an important investigational tool in many areas of analytical science, due to its high sensitivity and selectivity. It can be used to investigate real-time structure and dynamics both in solution state and under microscopes, particularly for bio-molecular systems.
How does it work?
Fluorescence occurs when a fluorescent capable material (a fluorophore) is excited into a higher electronic state by absorbing an incident photon and cannot return to the ground state except by emitting a photon. The emission usually occurs from the ground vibrational level of the excited electronic state and goes to an excited vibrational state of the ground electronic state. Thus fluorescence signals occur at longer wavelengths than absorbance. The energies and relative intensities of the fluorescence signals give information about structure and environments of the fluorophores.
The component parts necessary within a typical Fluorescence Spectrometer (Spectrofluorometer) are a sample holder, incident photon source (typically a xenon lamp), monochromators used for selecting particular incident wavelengths, focusing optics, photon-collecting detector (single, or preferably multiple channel) and finally a control software unit. An emission monochromator or cut-off filters are also usually employed. The detector is usually set at 90 degrees to the light source. The intrinsic sensitivity of fluorescence is also its biggest problem and care must be taken to record a true fluorescence signal of the analyte of interest.
A fluorescence emission spectrum is recorded when the excitation wavelength of light is held constant and the emission beam is scanned as a function of wavelength. An excitation spectrum is the opposite, whereby the emission light is held at a constant wavelength, and the excitation light is scanned as a function of wavelength. The excitation spectrum usually resembles the absorbance spectrum in shape.
Most materials are not naturally fluorescent. However, useful data, particularly in fluorescence microscopy can be obtained by staining non-fluorophores with an active label.
- Studies of molecular structures and molecular interactions
- Localization of molecules (esp. in biological systems) and in types of trace analysis.
- Changes in fluorescence intensity can be used to probe structural changes or binding of two molecules. The wavelength of tryptophan fluorescence can be used to determine whether a tryptophan is in an aqueous environment (longer wavelength) or buried deep within the protein (shorter wavelength).
- Fluorescence polarization anisotropy allows mobility of fluorophores to be studies.
- Sensitivity: Pico gram quantities of luminescent materials can be frequently studied.
- Selectivity: Deriving in part from the two characteristic wavelengths (excitation, fluorescence) of each compound.
- The variety of sampling methods available: dilute and concentrated suspensions and solid surfaces can all be readily studied and combinations of fluorescence spectroscopy and chromatography.
- With high pressure Xenon lamps which are still widely used as light sources.
- These lamps contain gas at several atmosphere pressure and thus should be handled with great circumspections (eye protection, gloves, chest protection recommended).
- Always operate in dust free environment with small temperature variation.
- Extreme precautions must be considered with regards to the cleanliness of cells. Fingerprints exhibit substantial fluorescence.
- Samples should be stored in clean glass vessels (not in plastic containers.