fluorescence

Examples of fluorescence in the following topics:

• Fluorescent Antibodies

  • Fluorescent antibodies are antibodies that have been tagged with a fluorescent compound to facilitate their detection in the laboratory.
  • Fluorescent molecules are used as substitutes for radioisotope or enzyme labels.
  • Fluorescent techniques are very specific and sensitive, so fluorescent antibody-based techniques require a fluorescent microscope.
  • Fluorescein fluoresces an intense apple-green color when excited under fluorescent microscopy.
  • Fluorescent antibody conjugates are commonly used in immunoassays.

• Fluorescence and Phosphorescence

  • Fluorescence and phosphorescence are photoluminescence processes in which material emits photons after excitation.
  • Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation.
  • Phosphorescence is a specific type of photoluminescence related to fluorescence.
  • Unlike fluorescence, a phosphorescent material does not immediately re-emit the radiation it absorbs.
  • Energy scheme used to explain the difference between fluorescence and phosphorescence

• Fluorescence Microscopy

  • Fluorescence microscopy is used to study specimens that are chemically manipulated to emit light.
  • This laboratory technique employs fluorescent dyes chemically linked to antibodies to help identify unknown microorganisms.
  • This method uses the specificity of an antibody to its antigen to deliver a fluorescent dye to a target molecule.
  • A filter is used to block the heat generated from the lamp and to match the fluorescent dye labeling the specimen.
  • Fixed endothelial cells stained with fluorescent dyes.

• Confocal Microscopy

  • Confocal microscopy is a non-invasive fluorescent imaging technique that uses lasers of various colors to scan across a specimen with the aid of scanning mirrors.
  • The biological sample to be studied is stained with antibodies chemically bound to fluorescent dyes similar to the method employed in fluorescence microscopy .
  • Unlike in conventional fluorescence microscopy where the fluorescence is emitted along the entire illuminated cone creating a hazy image, in confocal microscopy the pinhole is added to allow passing of light that comes from a specific focal point on the sample and not the other.

• Tracking Cells with Light

  • Advanced technology enables tracking cells with light by introducing fluorescent or luminescent reporter genes into the cells' genome.
  • Examples of such reporters are the genes encoding for Green Fluorescent Protein (GFP) and luciferase, respectively.
  • Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy , or bioluminesce (emit light) when luciferin, the substrate for luciferase is added.

• DNA Sequencing Based on Sanger Dideoxynucleotides

  • Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent dye.
  • The later development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
  • In dye-terminator sequencing, each of the four dideoxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths .
  • DNA sequencers carry out capillary electrophoresis for size separation, detection and recording of dye fluorescence, and data output as fluorescent peak trace chromatograms.
  • This is an example of the output of a Sanger sequencing read using fluorescently labelled dye-terminators.

• Interference Microscopy

  • There are three types of interference microscopy: classical, differential contrast, and fluorescence contrast.
  • Fluorescence differential interference contrast (FLIC) microscopy was developed by combining fluorescence microscopy with DIC to minimize the effects of photobleaching on fluorochromes bound to the stained specimen.
  • The same microscope is equipped to simulataneously image a specimen using DIC and fluorescence illumination.

• The FISH Technique

  • FISH is a hybridization technology which allows the labeling of target RNAs with a fluorescent probe.
  • FISH (fluorescence in situ hybridization) is a cytogenetic technique developed by biomedical researchers in the early 1980s.
  • FISH uses fluorescent probes bind to those targets that show a high degree of sequence complementarity.
  • Describe how fluorescent in situ hybridization (FISH) is used in clinical and biomedical studies to detect and localize the presence or absence of specific DNA sequences and to identify pathogens

• Reporter Fusions

  • Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins.
  • Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue light, the enzyme luciferase, which catalyzes a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed.
  • In the case of GFP which fluorescence one can deduce that the attached protein is wherever the fluorescence is.
  • As you can see the localization of the fused protein can now be determined using fluorescent reporter fusions.

• Purifying Proteins by Affinity Tag

  • Fluorescence tags are used to give visual readout on a protein.
  • GFP and its variants are the most commonly used fluorescence tags.
  • More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colorless if not).
  • Green fluorescent protein-tag, a protein which is spontaneously fluorescent and can be bound by nanobodies