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.