Fluorescence microscopy

(noun)

optical microscope that uses fluorescence to study properties of substances. A sample is illuminated with light of a wavelength that excites fluorescence in the sample. The fluoresced light, which is usually at a longer wavelength than the illumination, is then imaged through a microscope objective.

Related Terms

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.

  • Bacterial Chromosomes in the Nucleoid

    • The DNA-intercalating stains DAPI and ethidium bromide are widely used for fluorescence microscopy of nucleoids.

  • 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.

  • Scanned-Probe Microscopy

    • Scanned-probe microscopy uses a fine probe rather than a light-beam or electrons to scan the surface of a specimen and produce a 3D image.
    • Scanned-probe microscopy (SPM) produces highly magnified and three-dimensional-shaped images of specimens in real time.
    • Scanning tunneling microscopy (STM) measures a weak electrical current flowing between tip and sample as they are held apart.
    • Near-field scanning optical microscopy (NSOM) scans a very small light source very close to the sample.
    • Describe the different types of scanning probe techniques and their advantages over other types of microscopy