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 Microscope There 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

upright microscope light path

Further reading Olympus web resource ( Book "Fundamentals of light microscope and electronic imaging" by Douglas B. Murphy.