1.3.1 Fluorescence microscopy/-spectrocopy
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Module description: Is used for the measurement of single molecules in a living cell. Fluorophores are attached to the molcules of interest which are then subject of observation through high-sensitivity photo/video microscopy. This technique is useful for the identification of low copy number processes in the cell as well as for obtaining kinetics without perturbing the systems.
Fluorescence microscopy
The technique of fluorescence microscopy has become an essential tool in biology, biomedicine and in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorophores has made it possible to identify cells and sub-microscopic cellular components with a high degree of specificity amid non-fluorescing material capable of revealing the presence of a single molecule. A fluorescence microscope is a light microscope that illuminates specimen with the light of a specific wavelength causing the observed object to emit light with a different color due to absorbtion by the fluorophores. Fluorophores (such as green fluorescent protein (GFP), fluorescein or DyLight 488) are used in the specimen to label a component of interest to study properties of organic or inorganic substances. Life sciences widely use epifluorescence microscopes where excitation and observation of the fluorescence is from above (epi–) the specimen. These microscopes have become an important part in the field of biology, opening the doors for more advanced microscope designs such as: confocal microscope, total internal reflection fluorescence microscope (TIRF) and Vertico SMI combining localization microscopy with spatially modulated illumination uses standard fluorescence dyes and reaches an optical resolution below 10 nanometers (1 nanometer = 1 nm = 1 × 10−9 m). These microscopes combine the power of high performance optical components, such as laser, with computerized control of the instrument and digital image acquisition to achieve a level of sophistication that far exceeds that of simple observation by the human eye, collecting high-resolution 3-dimensional images of cells or other specimens. Microscopy now depends heavily on electronic imaging to rapidly acquire information at low light levels or at visually undetectable wavelengths.
Fluorescence spectroscopy
Fluorescence spectroscopy (FS) (fluorometry or spectrofluorometry), is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. FS measures emitted light, for example, by a labeled sample that has been excited by light at a shorter wavelength. The greater the difference in wavelength between the excitation and emission light the greater the potential for sensitive measurement of the fluorescence. The quantification of a dye is done with a Spectrofluorometer. Some protein or small molecules in cells are naturally fluorescent, this is called intrinsic or autofluorescence (such as NADH, tryptophan or endogenous Phycoerythrin or green fluorescent protein), alternatively specific or general protein, nucleic acids, lipids or small molecules can be "labelled" with a extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot. FS is an important investigational tool in many areas of analytical science, due to its extremely high sensitivity and selectivity. With many uses across a broad range of chemical, biochemical and medical research, it has become an essential investigational technique allowing detailed, real-time observation of the structure and dynamics of intact biological systems with extremely high resolution. It is particularly heavily used in the pharmaceutical industry where it has almost completely replaced radiochemical labeling. Time-resolved FS is usable when the fluorescent label has a long decay time. Light is collected at a time when non-specific fluorescence from the sample matrix and the micro plate, etc. has died down. The elimination of interfering background in this way makes a further big contribution towards sensitivity.
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