Fluorescence microscope

The fluorescence microscope is the most used microscope in the medical and biological fields. Fluorescence microscopy is used to study specimens that are chemically manipulated to emit light.These types of microscopes use high-powered light waves to provide unique image viewing options that are unavailable with traditional microscopes. In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near-ultraviolet) light source. Microorganisms appear as bright objects against a dark background. Fluorescence microscopy is used primarily in a procedure called fluorescent-antibody (FA) technique, or immunofluorescence.

How it works
The fluorescent microscope uses a high-pressure mercury, halogen, or xenon vapor lamp that emits a shorter wavelength than that emitted by traditional brightfield microscopy. These light sources produce ultraviolet light. When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color. Since ultraviolet light is used a larger amount of information can be gathered; thus, the resolution of the object increases.

Uses
Medical Sciences- A natural use for this technology is studying organisms that naturally have fluorescence characteristics. Environmental Sciences - Usually air and water samples are passed through membrane filters, and these filters are examined using fluorescence microscopy to determine if there is bacterial contamination.Industrial - There are many possible uses of fluorescence microscopy within a variety of industries. A very common example is the use of this technology within the food industry. You can directly count microbes in many food products and thus determine the contamination of the food in question. This method is far more sensitive than standard bacterial culturing.

Advantage
Fluorescence microscopy are highly sensitive and selective, Versatile (antibodies to label proteins, probes for nucleic acids, probes for a variety of cellular components, fluorescent proteins for live studies), potentially provide superb contrast because objects are self-luminous against a dark background.It has also the ability to separate absorbtion spectrum from emitted spectrum by virtue of Stokesshift and an extremely small number of fluorescent molecules (as few as 50 molecules per cubic micrometer) can be detected.

Disadvantages
Fluorescence detection sensitivity is compromised by background signals, either from endogenous sample constituents (autofluorescence) or from unbound or non-specific reagents. There are well-developed specimen preparation methods to manage and minimise background signal. There can also be blurring, bleaching, bleedthrough and autofluorescence.