In order to be more familiar and understand microscopic substance of GFP, Green Fluorescent Protein, One must understand and learn how fluorescence microscopy works and why it has become so important to modern biology, one must understand what the term fluorescence means. Fluorescence is the luminescence of a substance when it is excited by radiation. In microscopy, fluorescence ,it is said that it is used as a means of preparing specific biological probes. Some biological microscopic substances like chlorophyll and some oils and waxes have primary fluorescence with in them that may emit light and glow on dark environments. That process is known to be autofluoresce. But most microscopic biological molecules or structures do not fluorescence on their own, so they must be linked with fluorescent molecules or fluorochromes in order to create specific fluorescent probes and therefore can emit light with in their own. Scientist and researchers from north America concluded and believes that Fluorescence of a substance is seen when the molecule is exposed to a specific wavelength of light ,excitation wavelength or spectrum, and the light it emits ,the emission wavelength or spectrum, is always of a higher wavelength. To view this fluorescence in the microscope, several light filtering components are said to be needed. Such specific filters are significantly needed to isolate the excitation and emission wavelengths of a microscopic fluorochrome. However, a bright light source with proper wavelengths for excitation is also needed. For normal fluorescence applications as such, this is a mercury vapor arc burner. For fluorescence confocal microscope applications where up to 95 percent of the emission light is filtered out, specific wavelength lasers are used as these are extremely bright that the naked human eye. Mercury arc burners are very bright lamps with a limited lifetime and might require some maintenance and care to make sure that they are producing the brightest possible light beam for fluorescence excitation.
One other component is also said to be critically required that is a dichroic beam splitter or partial mirror which reflects lower wavelengths of light and allows higher wavelengths to pass. A beam splitter is important and is required because the objective acts as a condenser lens for the excitation wavelength as well as the objective lens for emission. One only wishes to see the light emitted from the microscopic fluorochrome and not any of the excitation light, and the beam splitter isolates the emitted light from the excitation wavelength. This epi-illumination type of light path is required to create a dark background so that the fluorescence can be easily seen by the naked human eye. The wavelength at which is seen to be a beam splitter allows the higher wavelengths to pass or transpose, must be set between the excitation and emission wavelengths of any given microscopic fluorochrome so that excitation light is reflected and emission of the light is allowed to pass through it where only a an electron microscope is best to equip in such research and experiments that involves fluorescence microscopy.
