Introduction to Fluorescence Microscopy
Fluorescence is a member of the ubiquitous luminescence family of processes in which susceptible molecules emit light from electronically excited states created by either a physical (for example, absorption of light), mechanical (friction), or chemical mechanism. Generation of luminescence through excitation of a molecule by ultraviolet or visible light photons is a phenomenon termed photoluminescence, which is formally divided into two categories, fluorescence and phosphorescence, depending upon the electronic configuration of the excited state and the emission pathway. Fluorescence is the property of some atoms and molecules to absorb light at a particular wavelength and to subsequently emit light of longer wavelength after a brief interval, termed the fluorescence lifetime. The process of phosphorescence occurs in a manner similar to fluorescence, but with a much longer excited state lifetime.
Derived from our introductory sections in the Physics of Light and Color, this section provides short explanations of the important associated phenomena as well as several tutorials and a references listing.
When coupled to the optical microscope, fluorescence enables investigators to study a phenomena in cellular biology. Foremost is the analysis of intracellular distribution of specific macromolecules in sub-cellular assemblies.
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Explore how electrons in fluorophores are excited from the ground state into higher energy states, and the events that occur as these molecules relax by photon emission to then fall back into the ground-level energy state.
Many environmental factors affect fluorescence emission such as interactions between the fluorophore and solvent molecules. Examine relaxation effects and spectral shifts that occur as a function of solvent polarity.
Photobleaching occurs when a fluorophore loses the ability to fluoresce due to photon-induced chemical damage. Explore variations in photobleaching rates in single, dual, and multiply labeled fluorescence specimens.
The field of fluorescence microscopy is experiencing a renaissance with the introduction of new techniques such as confocal, multiphoton, deconvolution, and total internal reflection, especially when coupled to advances in chromophore and fluorophore technology. The reference materials listed below were utilized in the construction of the fluorescence section of the Molecular Expressions Microscopy Primer.