Examples of Fluorescence microscopy in the following topics:
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- 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.
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- 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.
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- 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.
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- 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.
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- Serum can be directly used in agglutination, precipitation, complement fixation, fluorescent microscopy, and enzyme-linked assays.
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- 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.
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- 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
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- 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.
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- 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
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- 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.