Fluorescence | Fluorescence
Key Words: confocal, GFP, resolution, FRAP, FLIP, diascopic, episcopic, Fluorophore, spectral imaging, multidimensional imaging, FLIM, TIRF, fluorescence filters, signal-to-noise ratio, epi-fluorescence, PA-GFF, Kaede, quantum dots, laser tweezers, noise-terminator technology
Definition:Fluorescence is the phenomenon where absorption of light of a given wavelength by a molecule is followed by the emission of light at longer (visible) wavelengths
Fluorescence imaging uses high intensity illumination to excite fluorescent molecules in the sample. When a molecule absorbs photons, electrons are excited to a higher energy level. As electrons 'relax' back to the ground-state, vibrational energy is lost and, as a result, the emission spectrum is shifted to longer wavelengths.
Fluorescence emanates from the sample (and not the illuminating light). In epi-fluorescence microscopes, the objective both focuses the excitation light and collects light returning to the eyepiece or detector. Fluorescence is separated from excitation light by a dichroic mirror and appropriate filters: excitation light is reflected back into the objective while fluorescence is transmitted. Filters, excluding and / or transmitting selected wavelengths of light, optimize fluorescence and reduce unwanted 'background noise'.
Many substances auto-fluoresce and this has been exploited especially in botany, petrology, and semiconductor industry. Commercially available fluorophores, with well-defined excitation and emission spectra, can be used to 'stain' specific structures or molecules in a specimen. Judicious choice of fluorophores allows the identification of multiple targets as long as emission spectra can be cleanly separated and distinguished from auto-fluorescence.
Fluorescence imaging allows molecules beyond the resolution limit of the light microscope to be visualized. Fluorescence microscopy is a key technique in clinical diagnostic (for example, immunology, pathology, microbiology, cytogenetics) and research environments. Confocal fluorescence microscopy, in particular, has become an essential tool central to the study of structural and molecular dynamics in living cells. Fluorescence enables imaging techniques such as TIRF, FRET, FLIM, FLIP and FRAP. Fluorescence microscopy is also important for identifying fluorescent minerals, contaminants and impurities in materials science, geology, semiconductor inspection, and environmental protection.
Fluorescence imaging can be carried out 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. Inverted microscopes can be configured with laser based fluorescence applications such as PA-GFP, laser tweezers and FRAP. Resolution in fluorescence imaging is improved with the use of confocal microscopy (Nikon's eC1, C1, C1si and LiveScan Sweptfield systems). This eliminates light above and below the plane of focus to dramatically reduce blur. The C1si spectral imaging system enables separation of overlapping spectra. Nikon's Ti series inverted and Eclipse i-series upright microscopes include noise-terminator technology to improve signal-to-noise ratios. A number of specialized fluorescence objectives are available (Plan Fluor, Super Fluor, Plan Apochromat, Plan Apochromat VC). For industrial applications, Nikon recommends the Eclipse 90i and DIH-E systems.
The choice of fluorescence system will depend on the application. Consult Nikon for advice.
Stereo fluorescence microscopy: [microscopyu]