Fluophores

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Fluorophores

A fluorophore is a fluorescent chemical compound that can re-emit light upon light excitation. The fluorophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. The absorbed wavelengths and time before emission depend on both the fluorophore structure and its chemical environment, as the molecule in its excited state interacts with surrounding molecules. Excitation energies range from ultraviolet through the visible spectrum, and emission energies may continue from visible light into the near infrared region. Fluorescein, has been one of the most popularized fluorophores. From antibody labeling, the applications have spread to nucleic acids. Other common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine. Newer generations of fluorophores often perform better (more photostable, brighter, and less pH-sensitive). In medicine, the fluorophores are importants for the diagnose of some problems. Sometimes are used alone, as a tracer in fluids, as a dye for staining of certain structures, as a substrate of enzymes, or as a probe or indicator. Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods, for exemple, fluorescent imaging and spectroscopy.

Types of fluorophores

Organic dyes Synthetic organic dyes, such as fluorescein, were the first fluorescent compounds used in biological research. The small size of these fluors is a benefit over biological fluorophores for bioconjugation strategies because they can be crosslinked to macromolecules, such as antibodies, without interfering with proper biological function. Biological fluorophores The first use of a biological fluorophore for research applications occurred in the 1990s. Since that time many proteins have been designed for use in biological expression systems, and their use is now commonplace in biological research.

Quantum dots Quantum dots are nanocrystals with unique chemical properties that provide tight control over the spectral characteristics of the fluor. Quantum dots have been increasingly used in fluorescence applications in biological research. They are nanoscale-sized (2-50nm) semiconductors that, when excited, emit fluorescence at a wavelength based on the size of the particle; smaller quantum dots emit higher energy than large quantum dots, and therefore the emitted light shifts from blue to red as the size of the nanocrystal increases.

Vantages and disvantages

The benefit of the biological fluorophores is that expression plasmids can be introduced into either bacteria, cells, organs or whole organisms, to drive expression of that fluorophore either alone or fused to a protein of interest in the context of the biological processes studied. The use of fluorescent proteins can be time consuming, and expressing large amounts of light-producing proteins can cause reactive oxygen species and induce artifactual responses or toxicity. Additionally, the size of the fluorescent protein can change the normal biological function of the cellular protein to which the fluorophore is fused, and biological fluorophores do not typically provide the level of photostability and sensitivity offered by synthetic fluorescent dyes. Quantum dots have also been reported to be more photostable than other fluorophores. Additionally, quantum dots can be coated for use in different biological applications such as protein labeling. While the use of quantum dots in biological applications is increasing, there are reports of cell toxicity in response to the breakdown of the particles.