Fluorescein isothiocyanate
Fluorescein isothiocyanate is a derivative of fluorescein used in wide-ranging applications including flow cytometry. First described in 1942, FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group, replacing a hydrogen atom on the bottom ring of the structure. It is typically available as a mixture of isomers, fluorescein 5-isothiocyanate and fluorescein 6-isothiocyanate. FITC is reactive towards nucleophiles including amine and sulfhydryl groups on proteins. It was synthesized by Robert Seiwald and Joseph Burckhalter in 1958.
A succinimidyl-ester functional group attached to the fluorescein core, creating "NHS-fluorescein", forms another common amine reactive derivative that has much greater specificity toward primary amines in the presence of other nucleophiles.
FITC has excitation and emission spectrum peak wavelengths of approximately 495 nm/519 nm, giving it a green color. Like most fluorochromes, it is prone to photobleaching. Because of the problem with photobleaching, derivatives of fluorescein such as Alexa 488 and DyLight 488 have been tailored for various chemical and biological applications where greater photostability, higher fluorescence intensity, or different attachment groups are needed.
