Fluorescent Analogues in Biological Research


The information that can be obtained from a fluorescence measurement is influenced strongly by the spectral properties of the fluorophores. A large variety of probes is now available which bind spontaneously to macromolecules, react covalently with macromolecules, or display spectral changes in response to chemical species of interest.

Keywords: fluorophores; membrane probes; DNA probes; sensors

Figure 1.

Chemical structures of some natural or intrinsic fluorophores. Different derivatives of pyridoxyl display different emission spectra. NADH, the reduced form of nicotinamide–adenine dinucleotide, is fluorescent; NAD+ is not. In flavin–adenine dinucleotide (FAD), the oxidized form is fluorescent; the reduced form, FADH2, is not. In both NADH and FAD, the emission is partially quenched by the nearby adenine residue.

Figure 2.

Absorption (A) and fluorescence (F) spectra of tryptophan (TRP), reduced nicotinamide–adenine dinucleotide (NADH) and flavin–adenine dinucleotide (FAD).

Figure 3.

Chemical structures of commonly used extrinsic fluorophores. DNS‐Cl, dansyl chloride; FITC, fluorescein isothiocyanate; TRITC, tetraethylrhodamine isothiocyanate. Also shown are the excitation/emission wavelengths.

Figure 4.

Absorption (A) and fluorescence (F) spectra of fluorescein isothiocyanate (top), dansyl chloride (middle) and some cyanine dyes. The dashed line is the absorption spectrum of Cy3.

Figure 5.

Membrane probes and fluorescent lipid analogues. DPH, 1,6‐diphenyl‐1,3,5‐hexatriene; TMA, trimethylammonium.

Figure 6.

Probes that bind to deoxyribonucleic acid (DNA). Ethidium bromide intercalates between the base pairs of DNA. DAPI and TOTO bind to the minor groove of DNA. Also shown are the excitation/emission wavelengths of these probes.

Figure 7.

Fluorescent nucleic acid bases. Yt base is an unusual naturally occurring base found in an Escherichia coli transfer ribonucleic acid; 2‐aminopurine and isoxanthopterin are extrinsic fluorophores that are incorporated synthetically into deoxyribonucleic acid.

Figure 8.

Fluorescent analogues of nucleotides and oligonucleotides. Pyrene forms excimers with other nearby pyrene groups. Fluorescein is typically used as a resonance energy transfer donor.

Figure 9.

Fluorescent probes that are sensitive to chloride (a) or calcium (b). The emission of MQAE is quenched by diffusive collisions with chloride (values show the concentration of chloride, 0, 3, 10 mmol L−1. The absorption and emission of Fura‐2 displays spectral shifts upon binding calcium.

Figure 10.

Fluorogenic probes. These probes are used to detect the presence of enzymatic activity, typically in immunoassays or in studies of gene expression.

Figure 11.

A fluorogenic probe for human immunodeficiency virus (HIV) protease. The enzyme HIV protease releases the fluorophore (F) from the effects of a nearby acceptor (A). An increase in donor fluorescence is used to detect the HIV protease. FRET, fluorescence resonance energy transfer.

Figure 12.

Absorption (red) and fluorescence emission (blue) spectra of two representative phycobiliproteins, B‐phycoerythrin from the unicellular red alga Prophyridium cruentum, and R‐phycoerythrin from the red alga Gastroclonium coulteri.

Figure 13.

Long‐lifetime luminophores. (a) In transition metal complexes such as [Ru(bpy)2(dppz)]2+ the absorption and emission are due to a charge transfer transition of the entire complex. (b) In the terbium complex, the Tb is excited by resonance energy transfer from the coumarin. The emission occurs from the Tb atom itself. dppz is dipyrido[3,2‐a:2′,3′‐c]phenazine. TTHA‐cs 124 is the conjugate of triethylenetetraamine‐hexaacetic acid (TTHA) and 7‐amino‐4‐methyl‐2(1H)‐quinolinone (also called carbostyril 124 or cs 124)


Further Reading

Brand L and Johnson ML (1997) Fluorescence Spectroscopy. New York: Academic Press.

Haughland RP (1996) Handbook of Fluorescent Probes and Research Chemicals, 6th edn. Eugene, Oregon: Molecular Probes.

Hurtubise RJ (1990) Phosphorimetry: Theory, Instrumentation, and Applications. New York: VCH.

Lakowicz JR (1992–1997) Topics in Fluorescence Spectroscopy, vols I–V. New York: Plenum Press.

Wolfbeis OS (1991) Fiber Optic Chemical Sensors and Biosensors, vol. I. Boca Raton, Florida: CRC Press.

Wolfbeis OS (1991) Fiber Optic Chemical Sensors and Biosensors, vol. II. Boca Raton, Florida: CRC Press.

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Lakowicz, Joseph R(Apr 2001) Fluorescent Analogues in Biological Research. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002981]