Fluorescence microscopy Principle and applications.
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Transcript of Fluorescence microscopy Principle and applications.
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Fluorescence microscopy
Principle and applications
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Applications of fluorescence microscope in clinical samples?
• Fluorescent staining is commonly used to improve tuberculosis diagnosis efficiency as well as for malaria diagnosis
• Early detection of bacteria in blood cultures, and to detect and identify nucleic acids by color.
• Chromosomal anomalies ( FISH)
• Fluorescent antibodies provide a wide variety of immunologically specific, rapid diagnostic tests for infectious diseases.
can observe in live cells
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Principle of fluorescence
• When light radiation of high energy strikes a substance that can fluoresce, the substance absorbs that energy and converts a small part of it into energy (i.e. heat).
• The energy that is not absorbed by the substance is emitted again as light.
• The emitted light is called fluorescent light
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Primary and secondary fluorescence
• Substances that can be activated to fluoresce are called fluorochromes
• Fluorochromes may be naturally present in biological materials (Primary).
• or may be artificially introduced into these materials (secondary fluorescence)
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Fluorophores (Fluorochromes, chromophores)
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Basic fluorescence microscope parts• Compound microscope (basic platform and
lenses)
• High-power illuminator (excitation light)
• Exciter light filter (selects light color)
• A dichroic mirror further reflects the exciting light color, but it passes the
higher wavelength fluorescence light.
• Barrier filter (blocks low wavelength light).
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Fluorescent MicroscopeThere are two types of fluorescence
microscope:
• Transmitted light (excitation illumination through the specimen, usually from below)
• epi-illumination (incident light shining on the surface of the specimen)
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• The basic function of a fluorescence microscope is to irradiate the specimen with a specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light.
• Only the emission light should reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark (or black) background.
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Optical arrangement of transmitted fluorescence microscopy
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Optical arrangement of epi-illumination fluorescence microscopy
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Excitation / emission
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Excitation / emission
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Fluorescent Microscope.mp4
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Fluorescence Microscopy - Intracellular compartments.mp4
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upright microscope light path
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Further reading
Olympus web resource (http://www.olympusmicro.com)
Book"Fundamentals of light microscope and electronic imaging"
by Douglas B. Murphy.
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