Fluorescence in the life sciences

Fluorescence in the life sciences is used generally as a non-destructive way of tracking biological molecules by means of fluorescence. Some protein or small molecules in cells are naturally fluorescent, this is called autofluorescence (such as NADH, tryptophan or GFP), alternatively specific or general protein, nucleic acids, lipids or small molecules can be "labelled" with a fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. Additionaly a useful propriety is FRET where the energy is passed non-radiatively to a particular neighbouring dye, allowing proximity to be detected.

Comparison with radioactivity

Prior to its widespread use in the past three decades radioactivity was the most common label.

Advantages are:

• fluorescence is much safer and more convenient to use
• Several fluorescent molecules can be used simultaneously (given that they do not overlap, cf. FRET), whereas with radioactivity two isotopes can be used (tritium and a low energy isotope, eg. P-33 due to different intensities) but require special machinery (a tritium screen and a regular phosphor-imaging screen or a specific dual chanel detector(eg. [1]).

Note: a channel is similar to "colour", it is the pair of excitation and emission filters specific for a dye.

Disadvantages are:

• the dye may be a hindrance

Labels

Several organelle specific dyes are available[2] Green Fluorescent Protein (GFP), from the jellyfish Aequorea victoria, has become an extremely important research tool. GFP and related proteins are used as reporters for any number of biological events including such things as sub-cellular localization. Levels of gene expression are sometimes measured by linking a gene for GFP production to another gene, making a fusion protein.

Fluorophores can be attached to protein to specific functional groups, such as amino groups (succimidyl ester, isothiocyanide, hydrazine), carboxyl groups (carbodimide), thiol (maleimide, acetylbromide), azide (via click chemistry), or non-specificately (gluteraldehyde).

Methods

• Fluorescence microscopy of tissues, cells or subcellular structures is accomplished by labeling an antibody with a fluorophore and allowing the antibody to find its target antigen within the sample. Labeling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image.
• 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.

Ethidium bromide stained agarose gel. Ethidium bromide fluoresces orange when intercalating DNA and when exposed to UV 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 visualising the location of DNA fragments in agarose gel electrophoresis. Ethidium bromide can be toxic - a safer alternative is the dye SYBR Green.
• The DNA microarray
• Immunology: 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 the fluorescence.
• FACS (fluorescent-activated cell sorting)
• Fluorescence has been used to study the structure and conformations of DNA and proteins with techniques such as Fluorescence resonance energy transfer, which measures distance at the angstrom level. This is especially important in complexes of multiple biomolecules.

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. Bilirubin, 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 problems.

As of 2006, the number of fluorescence applications is growing in the biomedical, biological and related sciences. Methods of analysis in these fields are also growing, albeit with increasingly unfortunate nomenclature in the form of acronyms such as: FLIM, FLI, FLIP, CALI, FLIE, FRET, FRAP, FCS, PFRAP, smFRET, FIONA, FRIPS, SHREK, SHRIMP or TIRF. Most of these techniques rely on fluorescence microscopes. These microscopes use high intensity light sources, usually mercury or xenon lamps, LEDs, or lasers, to excite fluorescence in the samples under observation. Optical filters then separate excitation light from emitted fluorescence, to be detected by eye, or with a (CCD) camera or other light detectors (photomultiplier tubes, spectrographs, etc). Much research is underway to improve the capabilities of such microscopes, the fluorescent probes used, and the applications they are applied to. Of particular note are confocal microscopes, which use a pinhole to achieve optical sectioning – affording a quantitative, 3D view of the sample.