Organic compounds can be classified as aliphatic and aromatic compounds. Aliphatic compounds are open chain or branch compounds with long carbon chain. Aromatic compounds have unique aroma and well known due to extra stability of compound because of presence of delocalization of electrons. Aromatic compounds show different chemical and physical properties. They have wide range of applications in several industries. Many aromatic compounds are used as dyes in textile and leather industries.
Dyes are colored, ionizing and aromatic organic compounds that exhibit affinity towards the substrate to which it is being applied. Dyes are mostly applied in a aqueous solution and may or may not require a mordant to facilitate its binding on the material on which it is applied. Dyes are also used for identification of individual components of tissue sections in histological studies. Dyes can be classified on different basis as given below.
Fluorescent dye definition is given as non-protein molecules that are capable of absorption of light followed by reemission of light of longer wavelength. This light emission makes the dyes visible even if present in highly diluted concentrations. The fluorescent dyes are mostly used in the fluorescent labelling of biomolecules. These dyes may or may not be photostable but cannot be genetically encoded by genes.
Some common examples of fluorescent dye are listed below.
| Examples of Fluorescent dyes
|| 7-Amino-4-methylcoumarin pH 7.0
|| Indo-1 Ca2+
|| Cascade Blue
|| Amino Coumarin
|| Alexa 405
|| LysoSensor Blue
|| DyLight 405
|| 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0
|| Pacific Blue
|| Hoechst 33258-DNA
|| Marina Blue
|| 1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)
|| CFP (Cyan Fluorescent Protein)
|| 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS)
|| SYTO 45-DNA
|| Auramine O
|| Cy 2
|| LysoSensor Green
|| LysoSensor Green pH 5.0
|| Oregon Green 514 antibody conjugate pH 8.0
|| 6-Carboxyrhodamine 6G, hydrochloride
|| Erythrosin-5-isothiocyanate pH 9.0
|| 5-(and-6)-Carboxy-2', 7'-dichlorofluorescein pH 9.0
Fluorescent dyes are highly sensitive and selective to detect and identify the target with low toxicity. That is the reason they are widely used in biological research. We can even trace the presence of specific biomolecules in living cells with the help of these dyes. They are also used in cell culture and in vitro studies. In Fluorescence In Situ Hybridization which is also known as FISH single-dye labeled oligo-nucleotides acts as cytogenetic probes. The technique is used to detect the presence of specific DNA sequences on chromosomes. Single-dye labelled oligo-nucleotides also involve in detection of specific mRNAs in tissues. FISH process provides patterns of gene expression in living cells. Another use of labelled oligo-nucleotides is fluorescence-based sequencing. They are also one of the preferred choices to be used as probes for OLA systems that are oligo-nucleotide ligation assay.
Fluorescence resonance energy transfer (FRET) experiments are based on dual-dye labelled oligo-nucleotides. These experiments are mainly used for the determination of distances at intra- and intermolecular levels. Some highly specializing applications or research projects combine the fluorescent modifications with non-fluorescent modifications.
Fluorescent dyes can be categorised on the basis of color they emit. For example, propidium iodide and rhodamine are the red fluorescent dyes while fluorescein is a green fluorescent dye. These dyes are used in molecular studies. Fluorescent dyes of different colors are used to differentiate biomolecules of different origin or different biomolecules of same origin. For example, mouse specific antibodies are linked to green fluorescent dye called fluorescein whereas human-specific antibodies are linked to a red fluorescent dye, rhodamine.
The fluorescent chemical compounds that can re-emit light upon light excitation are known as fluorophore or fluorochrome. These compounds have aromatic groups and other planar or cyclic molecules. They have a large number of Ï€ bonds in their structures. These compounds can describe with the help of their
- absorption properties
- fluorescence properties
- spectral profile
- maximum wavelengths of absorbance and emission
- and also on fluorescence intensity.
In the excitation and emission spectra of a fluorochrome the percent transmission is plotted as a function of wavelength.
Usually fluorochromes have well-defined bands of excitation and emission. In the spectra the difference in wavelength between the peaks of these bands is called as Stokes shift. Here the emission intensity value varies with excitation wavelength. That proves that spectral distribution of emitted light does not depend on the excitation wavelength. As the environment changes like pH level, concentration, conjugation etc the excitation and emission bands can shift. Some dyes such as FURA-2 and Indo-1 have large shifts in their spectra with changes in environment. Photobleaching or fading can be defined as the photochemical reactions that cause the decrease in the fluorescence efficiency with time. Spectrofluorimeter is used to generate a spectrum that is combination of two spectrometers: an illuminating spectrometer and an analyzing spectrometer.