Fluorophores | Fluorophores

Key Words: photobleaching, phototoxicity, confocal, Fluorescence, FRAP, FLIP, FRET, FLIM, TIRF, epi-fluorescence

Definition:A molecule or part of a molecule that emits fluorescence following excitation with light

TECHNOLOGY:

When a photon excites a fluorophore, its electronic and vibrational energy levels are raised. When the fluorophore relaxes to the ground state, the fluorophore releases energy in the form of a photon. Energy is lost during the process, so the photon emitted by the fluorophore is of a lower energy (i.e. longer wavelength) that that absorbed (termed the Stokes shift or Stokes law). The intensity and wavelength of the emitted energy depend on both the fluorophore and its chemical environment.

There are a great many fluorophores commercially available for microscopic imaging (for example, GFPs, quantum dots, fluorescent dyes) and which differ in their excitation and emission characteristics. Fluorophores can also be described in terms of the efficiency of the fluorescence process i.e. their quantum yield (the ratio of the number of photons emitted to the number of photons absorbed) and by their fluorescence lifetime (the time the molecule stays in its excited state before emitting a photon).

Fluorophores may be targeted towards specific structures for imaging by transfection (for example in the case of GFPs in live cell imaging), by immuno-targeting, or by interaction of specific chemical groups with target groups in the specimen (for example, DAPI binds to DNA, and the isothiocyanate group in fluorescein isothiocyanate binds to amine groups on proteins).

APPLICATIONS:

Fluorophores and their resultant fluorescence provide an extremely powerful way of providing contrast in microscopic imaging, especially in biological environments. Several different cell structures and molecules can be simultaneously imaged using multiple fluorophores as long as their emissions or fluorescence lifetimes can be clearly distinguished. Fluorophores enable the tracking of single molecules using TIRF, and energy transfer between fluorophores can be exploited in identifying closely interacting molecules (FRET). Bleaching of fluorophores is exploited in techniques such as FLIP and FRAP to monitor molecular traffic in living cells. Fluorophores are also important in identifying minerals, contaminants and impurities in materials science, geology, semiconductor inspection, and environmental science applications.

MICROSCOPE CONFIGURATION:

Fluorescing fluorophores can be imaged on almost any Nikon upright, inverted, polarizing or stereo microscope with appropriate illumination (episcopic or diascopic) and filters. Exclusions include the Coolscope and SMZ6 stereo microscopes. Fluorescence imaging can also be carried out in Nikon's Biostation incubator imaging system. A number of specialized fluorescence objectives are available (Plan Fluor, Super Fluor, Plan Apochromat, Plan Apochromat VC)

SYSTEM SOLUTION:

For best possible fluorescence transmission from fluorophores Nikon's Ti inverted microscopes and i-series upright microscopes incorporate Noise-Terminator technology to improve signal-to-noise ratios.

LINKS:

Introduction to fluorescence: [microscopyu]