This microscope makes use of fluorescence and becomes a tool of inestimable value for scientific research, since it allows reaching high levels of sensitivity and microscopic resolution, allowing a different appreciation of the information that can be obtained from the specimens and that is generally goes unnoticed.
Fluorescence is a luminescence phenomenon that was initially observed by Sir George Stokes in 1852, and later physically explained in 1935 by Alexander Jablonski. It is the property that certain chemical elements called fluorophores or fluorochromes have of emitting visible light when intense radiation strikes them; in other words, they absorb light of a certain wavelength (for example ultraviolet light or blue monochromatic light) and then emit light of a longer wavelength (of a certain color, green, red, yellow). It is a short-lived luminescence phenomenon, emitted simultaneously with the excitation.
Requirements for the fluorescence microscope
• Light source: An intense light source is needed to excite fluorescence in the specific spectrum of each fluorochrome. It must be taken into account that fluorescence is temporary and lighting produces a photobleaching effect on the fluorochrome; furthermore, living cells can be damaged by the intense radiation. The light must be of a short wavelength. High-pressure mercury lamps are used that work differently from incandescent filament lamps. Ultraviolet light and laser beams are also used. Many models work with epi-lighting.
• Filters: They are those that allow the passage of light of a certain wavelength, the range and color necessary to excite the fluorochrome and block unwanted lengths. Once filtered, the light falls on the specimen by reflection from a dichroic mirror (epi-illumination) and is filtered again to be observed.
• Objectives: They must have a great capacity to transmit light and provide a high quality image. In the same way they must have a large numerical opening.
Fluorescence microscope applications
There are numerous applications of fluorescence microscopy, notably in biology and medicine:
• Labeling of molecules in cells and tissues for their characterization and identification.
• Study of normal and pathological cells.
• Immunological studies.
• Mineralogy.