Fluorescence microscopy is a rapid expanding technique, both in the medical and biological sciences. The technique has made it possible to identify cells and cellular components with a high degree of specificity. The absorption and subsequent re-radiation of light by organic and inorganic specimens is typically the result of well-established physical phenomena described as being either fluorescence or phosphorescence. Certain antibodies and disease conditions or impurities in inorganic material can be studied with the fluorescence microscopy.
The technique of fluorescence microscopy has become an essential tool in biology and the biomedical sciences, as well as in materials science due to attributes that are not readily available in other contrast modes with traditional optical microscopy.
A variety of specimens exhibit auto fluorescence when they are irradiated, a phenomenon that has been thoroughly exploited in the fields of biology. Fluorochromes are stains that attach themselves to visible or sub-visible structures, are often highly specific in their attachment targeting, and have a significant quantum yield. In analogy to a Chromophore, it is a component of a molecule which causes the molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore. This technology has particular importance in the field of biochemistry and protein studies, e.g. in immunofluorescence and immunohistochemistry. Fluorescein isothiocyanate, a reactive derivative of fluorescein, has been one of the most common fluorophore chemically attached to other, non-fluorescent molecules to create new and fluorescent molecules for a variety of applications. Other historically common fluorophore are derivatives of Rhoda mine, coumarin and cyanine.
Fluorescence is caused by the following sequence
1. Energy is absorbed by the atom which becomes excited.
2. The electron jumps to a higher energy level.
3. Soon, the electron drops back to the ground state, emitting a photon, the atom fluorescence.
It is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption. The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. The technique is used to study specimens, which can be made to fluoresce. The sample can either be fluorescing in its natural form like chlorophyll and some minerals, or treated with fluorescing chemicals.
How it works?
The basic function of a fluorescence microscope is to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light. In a properly configured microscope, 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 background. The limits of detection are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence.
In order to generate sufficient excitation light intensity to produce detectable emission, powerful compact light sources, such as high-energy short arc-discharge lamps, are necessary. The most common lamps are mercury burners, ranging in wattage from 50 to 200 Watts, and the xenon burners that range from 75 to 150 Watts. These light sources are usually powered by an external direct current supply, furnishing enough start-up power to ignite the burner through ionization of the gaseous vapor and to keep it burning with a minimum of flicker.