Date post: | 14-Dec-2015 |
Category: |
Documents |
Upload: | roger-jenkins |
View: | 281 times |
Download: | 2 times |
Fluorescence microscopy
Principle and applications
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
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
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)
Fluorophores (Fluorochromes, chromophores)
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).
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)
• 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.
Optical arrangement of transmitted fluorescence microscopy
Optical arrangement of epi-illumination fluorescence microscopy
Excitation / emission
Excitation / emission
Fluorescent Microscope.mp4
Fluorescence Microscopy - Intracellular compartments.mp4
upright microscope light path
Further reading
Olympus web resource (http://www.olympusmicro.com)
Book"Fundamentals of light microscope and electronic imaging"
by Douglas B. Murphy.