One branch of microscopy, as the name implies, deals with the study of a sample through manipulating the light source itself. Fluorescence Microscopy is a technique used to study specimens that have the characteristics of being fluorescent or can be made fluorescent. The main idea behind this technique is based on the light phenomenon wherein certain materials emits energy detectable as visible light when irradiated with a certain wavelength of light source. Usually, the components of an observed specimen are specifically labeled with a fluorescent molecule called a fluorophore causing the target to emit longer wavelengths of light of a different color than the rest. This can be attained by either treating the specimen with fluorenscing chemicals or stimuli, or in some cases the specimen can be fluorenscing by itself in its natural form such as chlorophyll and other known minerals. With the utilization of an emission filter, the illumination light can then be sorted out from the rest of the weaker emitted wavelength of fluorescent light.

It is important that the illumination light is carefully focused on the sample in order to excite the fluorescent species or materials within the observed object. The illuminating light is also regarded by some as the ‘excitation light’. The technique can be explained or viewed in many ways but can be achieved with the use of a conventional microscope with some effort and careful study of the behavior of light.

More…An excitation-emission configuration wherein the two lights, discussed as the excitation and emission light, traveling through the objective is better known as epifluorescence. It is important within this process to split the illumination or excitation light from the fluorescent emission light that comes from the sample. The background illumination can sometimes be excessive if not carefully adjusted which can also degrade the overall image. Other factors that can affect the image quality are color “noise” and poor contrast.These are just among the basic theories and concepts behind fluorescent microscopy. Most of the concept within is entirely based on the behavioral structure and characteristics of light and the separation of colors. Read more on this subject