Fluorescence

Principle

Fluorescence, as other types of luminescence, is mostly found as an optical phenomenon in cold bodies (in contrast to incandescence, a process with a flame), in which a molecule absorbs a high-energy photon, and re-emits it as a lower-energy photon with a longer wavelength (λ). The energy difference between the absorbed and emitted photons ends up as molecular vibrations, finally in the form of heat. Usually the absorbed photon is in the UV, and the emitted light is in the visible range, but this depends on the absorbance curve and the shift to higher emitted λ (Stokes shift: Δλ) of the particular fluorophore (the molecule with the fluorescent structure).

The process can be described by:

S₁ → S₂ + h/λ_emitted.

The system starts in state S₁, and after the fluorescent emission of a photon with energy hν, it is in state S₂ where h is Planck’s quantum mechanical constant, being 6.626∙10^-34 Js.

Applications

There are many natural and synthetic compounds that exhibit fluorescence, and they have a number of medical, biochemical and industrial applications (fluorescent lighting tubes).

The fluorophore attached by a chemical reaction to bio-molecules enables very sensitive detection of the molecule.

Examples are:

Automated sequencing of DNA by the chain termination method; each of four different chain terminating bases has its own specific fluorescent tag. As the labeled DNA molecules are separated, the fluorescent label is excited by a UV source, and the identity of the base terminating the molecule is identified by the wavelength of the emitted light.

DNA detection The compound ethidium bromide, when free to change its conformation in solution, has very little fluorescence. Ethidium bromide's fluorescence is greatly enhanced when it binds to DNA, so this compound is very useful in visualizing the location of DNA fragments in agarose gel electrophoresis (see Electrophoresis).

The DNA microarray.

Immunology and immonohistochemistry An antibody has a fluorescent chemical group attached, and the sites (e.g., on a microscopic specimen) where the antibody has bound can be seen, and even quantified, by fluorescence.

FACS, fluorescent-activated cell sorting.

Fluorescence resonance energy transfer and similar techniques has been used to study the structure and conformations of DNA and proteins. This is especially important in complexes of multiple biomolecules.

Calcium imaging Aequorin, from the jellyfish Aequorea victoria, produces a blue glow in the presence of Ca²⁺ ions (by a chemical reaction). Other fluorescent dyes are calcium orange and the intracellular indicator Indo-1. It has been used to image calcium flow in cells in real time, especially in neurobiological applications. It has a long history in research of hippocampus slices. Imaging at the light microscopic and confocal level (see Confocal microscopy) is also used to explore the contribution of inward calcium currents and calcium release in relation to synaptic transmission in neurons. Specific applications are analyses of neuronal networks and synaptic plasticity, often studied with the patch-clamp technique and voltage clamp technique. This techniques may use the voltage sensitive Ca²⁺ dyes Fluo, Ca-green en Fura.

More Info

The success with aequorin has led to the discovery of Green Fluorescent Protein (GFP), an important research tool. GFP and related proteins are used as reporters for any number of biological events including sub-cellular localization. Levels of gene expression are sometimes measured by linking a gene for GFP production to another gene. Fluorescent calcium indicator proteins [FCIPs]) are Ca²⁺-sensitive GFP varians. Fig. 1 shows an example of light-evoked Ca²⁺ responses in retinal ganglion cells.

Fig. 1. Intact, light-sensitive retinal whole mount. A Blood vessels are red and active retinal ganglion cells are green (FCIP-positive). B Light-stimulus-evoked Ca²⁺ response (black trace; gray traces are single trials) measured in the soma with F/F the relative fluorescence changes. After PLoS Biol. 2004; 2(6): e163.

Also, many biological molecules have an intrinsic fluorescence that can sometimes be used without the need to attach a chemical tag. Sometimes this intrinsic fluorescence changes when the molecule is in a specific environment, so the distribution or binding of the molecule can be measured. Biliburin, for instance, is highly fluorescent when bound to a specific site on serum albumin. Zinc protoporphyrin, formed in developing red blood cells instead of hemoglobin when iron is unavailable or lead is present, has a bright fluorescence and can be used to detect these abnormality.

Source: Wikipedia