Fluorescence Microscopy

Microscopy Methods in Microbiology
Microroscopy Methods in Microbiology Part 2
Microroscopy Methods in Microbiology - Introduction
Observation of Microbes in the Living State
Observation of Microbes in Smears
Dyes and Staining
Useful pH Range and Colour Change of Some Indicator Dyes
Factors Affecting Staining
Microscopy and Micrometry
Microscopy and Micrometry - Introduction
Behaviour of Light
Light Theories
Resolving Power
Refraction and Reflection
Bright Field Microscopy
Use of Bright Field Microscope

Fluorescence Microscopy

	Fluorescence Microscopy Fluorescence microscopy is very useful in gaining structural details of the specimen. When certain materials like some dyes, uranium ore, uranium glass, oil droplets etc. are exposed to ultra vi let light, they absorb the energy of ultra violet light and emits it in a form of a visible light. The substances are said to fluoresce. Dyes like acridine orange-R, auramine-O, primulin, thioflavin S, thiazo yellow-G, morin, etc. are used to stain bacteria and examined under the ultra violet microscope, they arc seen as bright objects against a dark background. Dyes having specificity for type of bacteria are useful in diagnosis, for example auramine-O is used to detect Mycobacterium tuberculosis from smear or sputum.
Fluorescent antibody technique is the modification of above technique and was introduced by Coons et al(6) in the field of immunology. The principle is that the fluorescent dyes like fluorescein isothiocyanate (FITC) and lissamine rhodamine-B (RB 200) are tagged to the proteins, including serum antibodies, without alt ration or interference with the biological or immunological properties, of the proteins. These proteins can be easily seen under the fluorescent microscope.
In microbiology, this technique is commonly employed by using serum globulin conjugated with fluorochrome to locate its specific antigen This technique has wide applications in bacteriology, virology, parasitology, mycology and immunology often as a parallel technique to the traditional serological procedures in diagnosis. The method for the detection or identification of unknown ,bacteria, using cell specific fluorescent antibody is schematically described. For further details readers are requested to see the articles by Nairn (7) and Walker, Batty and Thomson (8)
Diagram showing the mechanism of fluorescent antibody technique. The bacterial cell coated with fluorescent dye-antibody conjugate will glow brilliantly when viewed under fluorescent microscope. Microscope suitable for this purpose are readily available now, but it is possible to convert bright-field microscope for fluorescent work. The main object of this instrument is to transmit as much as possible the fluorescence emitted by the object, since this fluorescence is of low intensity. To avoid loss of intensity, monocular microscope is preferred.
	At the same time the ultra violet light (peak value 365 nm) used for this purpose should be available to the object with maximum intensity. Therefore , the substage condenser should be made of ultra violet transmitting glass. The objectives must have highest N.A. and they should be non fluorescent. When fluorescent is used to defect the organism they appear larger than the normal. As a result, low power objective can be effectively. This facilitates the inspection of larger area of specimen in shorter time.

The use of oil-immersion objective poses a problem because usual immersion oils become fluorescent after exposure to air and more particularly to ultra light. Analar grade glycerol is useful, but not the best, since it does not have the ideal refractive index. Therefore, it is better to use highest available magnification with dry objectiv

Two filter systems are necessary in this microscopy. Primary or exciter filter is placed in between the illumination source and the objective. This filter is usually blue and allows ultra violet light to pass. Secondary or barrier filter is placed in the eyepiece or anywhere between eye and object. It allows only passage of light from fluorescing specimen. It depends upon the type of dye used for the technique.

Home | Site map | Submit Article | Directory | Search