Fluorescence Microscope

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Fluorescence Microscope

Fluorescence Microscope

Fluorescence microscopy is based on the property of certain compounds known as fluorochromes which exhibit the phenomenon of fluorescence under ultraviolet (UV) light.

These compounds have the capacity to absorb light from the short visible to long UV portion of the spectrum depending upon the fluorochrome used and re-emit the absorbed energy in the form

of longer wavelength visible light. Acridine orange and auramine are fluorochromes which can be used to stain bacteria directly. Indirect usage of the technique depends on conjugating fluorochromes which as fluorescein isothiocyanate (FITC) which conjugate with antibodies resulting in the formation of fluorochrome conjugated antibodies.

This technique know as immunofluorescence is sensitive as well as specific for detection and identification with a fluorochrome.

Operation of dark field

Operation of dark field condenser to achieve dark field microscopy. The annular aperture in the condenser focuses a hollow cone of light onto the specimen. Only rays scattered by the specimen (dashed lines) enter the objective to form an image

1. Intermediate image plane	2. Objective lens
3. Specimen plane	4. Condenser
5. Annular aperture	6. Source

This is done by treating the suspected sample on a slide with the tagged antibodies. Under a flourescence microscope the bacterial cells in question, if present, will bind with the tagged antibodies and begin to fluoresce.

Light from high intensity mercury arc or xenon lamps that are designed to give illumination of desired intensity and wavelength is passed through an exciter filter and a dichromatic beam splitter.

The exciter filter limits transmitted wavelengths to a narrow band to suit the type of fluorochrome used and the dichromatic beam splitter directs exciting wavelength to the objective and begin their passage in the reverse direction once again through the beam splitter to reach the barrier filter.

The barrier filter is intended to remove wavelengths of light not caused by fluorescence and transmit the light to the ocular piece to form an image that can be visualised or photographed.

In recent times, a narrow beam of laser light is used to excite fluorescence to obtain three dimensional pictures of fluorochrome stained bacteria. The microscope designed to utilise this technique is know as confocal microscope.

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