Brief Overview of Fluorescence
The phenomenon of fluorescence was known by the middle of the nineteenth century. British scientist Sir George G. Stokes first made the observation that the mineral fluorspar exhibits fluorescence when illuminated with ultraviolet light, and he coined the word "fluorescence". Stokes observed that the fluorescing light has longer wavelengths than the excitation light, a phenomenon that has become to be known as the Stokes shift. Fluorescence microscopy is an excellent method of studying material that can be made to fluoresce, either in its natural form (termed primary or autofluorescence) or when treated with chemicals capable of fluorescing (known as secondary fluorescence). The fluorescence microscope was devised in the early part of the twentieth century by August Köhler, Carl Reichert, and Heinrich Lehmann, among others. However, the potential of this instrument was not realized for several decades, and fluorescence microscopy is now an important (and perhaps indispensable) tool in cellular biology.
Fluorescence microscopy is a rapidly expanding and invaluable tool of investigation. Its advantages are based upon attributes not as readily available in other optical microscopy techniques.
To achieve maximum fluorescence intensity, the fluorochrome is excited at the wavelength at the peak of the excitation curve, and the emission is selected at the peak wavelength of the emission curve.
See how photon energy is absorbed by an electron to elevate it into a higher energy level and how the energy can be released, in the form of a lower energy photon, when the electron falls back to the original ground state.
Examine how electrons in fluorophores are excited from the ground state into higher electronic energy states and the events that occur as these excited molecules emit photons and fall back into lower energy states.
Explore the spectral overlap regions of fluorescence filter combinations, and how changes to the filter properties help determine the bandwidth of wavelengths passed through the various filter sets.
Selected Literature References
The reference materials listed below were utilized in the construction of the introductory fluorescence section in the Molecular Expressions Microscopy Primer.