Fluorescence Microscopy (A Type of microscope )

Fluorescence Microscopy

A substance is said to be fluorescent when it absorbs the energy of short light waves, such as blue of Vireo cholera and Campylobacter species.

Problems in the use of dark-ground microscope Problems are frequently encountered in the use of dark-ground microscopes. 

These include: 

1. Difficulty in focusing or centering of the dark-ground condenser.

2. Using slides and covers lips that are not completely clean.

3. Examining too dense a preparation.

4. The presence of air bubble in the immersion oil.

5. Insufficient oil contact between the slide and the condenser.

Fluorescence Microscopy A substance is said to be fluorescent when it absorbs the energy of short light waves, such as bluelight, and releases or emits the light of a longer wavelength, such as green or red light. This phenomenon is called fluorescence. It is being increasingly used in clinical laboratories because it can be adapted to rapid tests for the detection of microorganisms, antibodies and many other substances. When fluorescence microscopy is used for the detection of antigen-antibody reaction, it is known as immunofluorescence. 

Principle In fluorescnce microscopy, the "invisible" ultraviolet light is used to “illuminate" particles or microorganisms which have been previously treated with fluorescing dyes known as fluorochromes. 

The fluorescent dye absorbs the invisible UV light of shorter wavelength and emits light of longer wavelength which is within the visible spectrum of light. Thus the background is dark because there is no visible light, and only the specimen (cells, microorganisms, etc.) stained with a fluorescent dye appears as a bright object. 

An example of a commonly used fluorescent dye in immunofluorescence is fluorescin isothiocynate (FIT) which gives green fluorescence.

The light path of ultraviolet rays first passes through an optical filter, called exciter filter, which removes other non-specific wavelengths (colours) of light and allows only the UV rays. Using an immersion dark-ground condenser, the light is directed obliquely onto the object. 

These rays then pass through the specimen stained with a fluorochrome. The fluorochrome will absorb the UV light and emit visible light, making the object visible in its characteristic colour. A yellow barrier filter is fixed just above the objectives to prevent dangerous UV rays from entering the eye of the observer. 

A recent development in fluorescence microscopy is the use of reflected light and dichroic filters which has resulted in better illumination. Figure 3.25 shows the essential parts of a fluorescence microscope.

Setting up fluorescence microscope Fluorescence microscope is available as a complete unit and can be set up using manufacturer's instructions. However, an ordinary light microscope can be converted into a fluorescence microscope with some accesories as follows: 

1. Set up the expensive mercury vapour lamp (which is very bulky and clumsy to use) or relatively smaller and cheaper quartz iodine lamp in front of the mirror.

2. Fix the dark ground condenser in its place;

and focus and centre the condenser with the aid of a non-fluorescing immersion fluid such as liquid paraffin.

3. Insert the primary exciter filter in the filter holder in the sub stage condenser.

4. Insert the barrier filter above the objective, but below the eyepiece.

5. Place the specimen stained with the appropriate fluorochrome on the stage and examine. A successful fluorescence microscopy very much depends on experience.

Some applications of fluorescence microscopy 

1. Detection of acid-fast bacilli (AFB) in sputum or cerebrospinal fluid (CSF) whenstained with auramine fluorescent dye.

2. Examination of acridine-orange stained specimens for the detection of Trichomonas vaginalis, intracellular gonococci and meningococci, and other parasites and microorganisms.

3. In immunodiagnostic, using both direct and indirect antibody techniques. It is very useful with monoclonal reagents because of the high specificity.