Fluorescein isothiocyanate
Encyclopedia
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein
used in wide-ranging applications including flow cytometry
. FITC is the original fluorescein molecule functionalized
with an isothiocyanate
reactive group (-N=C=S), replacing a hydrogen
atom on the bottom ring of the structure. This derivative is reactive towards nucleophiles including amine
and sulfhydryl groups on protein
s.
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/521 nm. 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.
FITC protein labeling (e.g. antibodies) implicates photometric determination of the labeling efficiency with minimal sample amounts. Specialized nano-volume photometer offer the possibility to determine protein concentration as well as incorporation of FITC with submicroliter volumes (starting with 0.3 µl).
Fluorescein
Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....
used in wide-ranging applications including flow cytometry
Flow cytometry
Flow cytometry is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical...
. FITC is the original fluorescein molecule functionalized
Functional group
In organic chemistry, functional groups are specific groups of atoms within molecules that are responsible for the characteristic chemical reactions of those molecules. The same functional group will undergo the same or similar chemical reaction regardless of the size of the molecule it is a part of...
with an isothiocyanate
Isothiocyanate
Isothiocyanate is the chemical group –N=C=S, formed by substituting sulfur for oxygen in the isocyanate group. Many natural isothiocyanates from plants are produced by enzymatic conversion of metabolites called glucosinolates. These natural isothiocyanates, such as allyl isothiocyanate, are also...
reactive group (-N=C=S), replacing a hydrogen
Hydrogen
Hydrogen is the chemical element with atomic number 1. It is represented by the symbol H. With an average atomic weight of , hydrogen is the lightest and most abundant chemical element, constituting roughly 75% of the Universe's chemical elemental mass. Stars in the main sequence are mainly...
atom on the bottom ring of the structure. This derivative is reactive towards nucleophiles including amine
Amine
Amines are organic compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are derivatives of ammonia, wherein one or more hydrogen atoms have been replaced by a substituent such as an alkyl or aryl group. Important amines include amino acids, biogenic amines,...
and sulfhydryl groups on protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s.
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
Emission spectrum
The emission spectrum of a chemical element or chemical compound is the spectrum of frequencies of electromagnetic radiation emitted by the element's atoms or the compound's molecules when they are returned to a lower energy state....
peak wavelengths of approximately 495 nm/521 nm. Like most fluorochromes, it is prone to photobleaching
Photobleaching
Photobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing...
. Because of the problem with photobleaching, derivatives of fluorescein such as Alexa 488
Alexa (fluor)
The Alexa Fluor family of fluorescent dyes is produced by Molecular Probes, a subsidiary of Invitrogen. Alexa Fluor dyes are typically used as cell and tissue labels in fluorescence microscopy and cell biology....
and DyLight 488
DyLight Fluor
The DyLight Fluor family of fluorescent dyes are produced by Dyomics in collaboration with Thermo Fisher Scientific. DyLight dyes are typically used in biotechnology and research applications as biomolecule, cell and tissue labels for fluorescence microscopy, cell biology or molecular...
have been tailored for various chemical and biological applications where greater photostability
Photobleaching
Photobleaching is the photochemical destruction of a fluorophore. In microscopy, photobleaching may complicate the observation of fluorescent molecules, since they will eventually be destroyed by the light exposure necessary to stimulate them into fluorescing...
, higher fluorescence intensity, or different attachment groups are needed.
FITC protein labeling (e.g. antibodies) implicates photometric determination of the labeling efficiency with minimal sample amounts. Specialized nano-volume photometer offer the possibility to determine protein concentration as well as incorporation of FITC with submicroliter volumes (starting with 0.3 µl).