Fluorescence microscopy

Examples of Fluorescence microscopy in the following topics:

• Fluorescence Microscopy

  • Fluorescence microscopy is used to study specimens that are chemically manipulated to emit light.
  • The fluorescent microscope uses a high-pressure mercury, halogen, or xenon vapor lamp that emits a shorter wavelength than that emitted by traditional brightfield microscopy.
  • This method uses the specificity of an antibody to its antigen to deliver a fluorescent dye to a target molecule.
  • Fluorescence microscopy does not allow examination of live microorganisms as it requires them to be fixed and permeabilized for the antibody to penetrate inside the cells.
  • 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.
  • Confocal microscopy has multiple applications in microbiology such as the study of biofilms and antibiotic-resistant strains of bacteria.
  • Tetrahymena cell, visualized using GFP-labeled anti-beta tubulin antibodies under confocal microscopy.

• Fluorescent Antibodies

  • Fluorescent antibodies are antibodies that have been tagged with a fluorescent compound to facilitate their detection in the laboratory.
  • 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.
  • The chemical manipulation in labeling antibodies with fluorescent dyes to permit detection by direct microscopy examination does not impair antibody activity.
  • Fluorescent antibody conjugates are commonly used in immunoassays.

• Interference Microscopy

  • Interference microscopy is a variation of phase-contrast microscopy that uses a prism to split a light beam in two.
  • Interference microscopy uses a prism to split light into two slightly diverging beams that then pass through the specimen.
  • 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.

• Cultivation of Specimen

  • Serum can be directly used in agglutination, precipitation, complement fixation, fluorescent microscopy, and enzyme-linked assays.

• Tracking Cells with Light

  • Advanced technology enables tracking cells with light by introducing fluorescent or luminescent reporter genes into the cells' genome.
  • The most commonly used reporter genes have biofluorescent or bioluminescent characteristics and can be visualized with the aid of microscopy and other non-invasive imaging equipments.
  • 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.

• Prochlorophytes

  • Picoplankton observed by epifluorescence microscopy, a technique which allows the detection of certain groups of cells possessing fluorescent pigments; and example would be Synechococcus, which possess phycoerythrin

• 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.

• 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

• Electron Microscopes

  • The original form of electron microscopy, transmission electron microscopy, works in a similar manner using electrons.
  • The spatial variation in this information (the "image") is then magnified by a series of magnetic lenses and it is recorded by hitting a fluorescent screen, photographic plate, or light-sensitive sensor such as a CCD (charge-coupled device) camera.