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.

Brief Overview of Fluorescence - Derived from our introductory sections in the Physics of Light and Color portion of the Microscopy Primer, this section provides short explanations of the important associated phenomena as well as several interactive Java tutorials and a references listing.

Basic Concepts in Fluorescence - When coupled to the optical microscope, fluorescence enables investigators to study a wide spectrum of phenomena in cellular biology. Foremost is the analysis of intracellular distribution of specific macromolecules in sub-cellular assemblies, such as the nucleus, membranes, cytoskeletal filaments, mitochondria, Golgi apparatus, and endoplasmic reticulum. In addition to steady state observations of cellular anatomy, fluorescence is also useful to probe intracellular dynamics and the interactions between various macromolecules, including diffusion, binding constants, enzymatic reaction rates, and a variety of reaction mechanisms, in time-resolved measurements. Other important processes are also targets for investigation using the high degree of specificity and spatial resolution available with fluorescence microscopy. For example, fluorescent probes have been employed to monitor intracellular pH and the localized concentration of important ions, and for the study of cell viability and the factors that influence the rate of apoptosis. Likewise, important cellular functions such as endocytosis, exocytosis, signal transduction, and transmembrane potential generation have come under study with fluorescence microscopy. In reviewing the large number of applications that benefit from fluorescence analysis, it is apparent why the significant utility of fluorescence microscopy has driven this technique to the forefront of biomedical research.

Jablonski Energy Diagram - Absorption of energy by fluorochromes occurs between the closely spaced vibrational and rotational energy levels of the excited states in different molecular orbitals. The various energy levels involved in the absorption and emission of light by a fluorophore are classically presented by a Jablonski energy diagram, named in honor of the Polish physicist Professor Alexander Jablonski. This tutorial explores how electrons in common fluorophores are excited from the ground state into higher electronic energy states, and the events that occur as these excited molecules relax by photon emission and other mechanisms to ultimately fall back into the ground-level energy state.

Solvent Effects on Fluorescence Emission - A variety of environmental factors affect fluorescence emission, including interactions between the fluorophore and surrounding solvent molecules (dictated by solvent polarity), other dissolved inorganic and organic compounds, temperature, pH, and the localized concentration of the fluorescent species. The effects of these parameters vary widely from one fluorophore to another, but the absorption and emission spectra, as well as quantum yields, can be heavily influenced by environmental variables. In fact, the high degree of sensitivity in fluorescence is primarily due to interactions that occur in the local environment during the excited state lifetime. This interactive tutorial explores relaxation effects and associated spectral shifts that occur as a function of solvent polarity.

Photobleaching - The phenomenon of photobleaching (also commonly referred to as fading) occurs when a fluorophore permanently loses the ability to fluoresce due to photon-induced chemical damage and covalent modification. Upon transition from an excited singlet state to the excited triplet state, fluorophores may interact with another molecule to produce irreversible covalent modifications. The triplet state is relatively long-lived with respect to the singlet state, thus allowing excited molecules a much longer timeframe to undergo chemical reactions with components in the environment. The average number of excitation and emission cycles that occur for a particular fluorophore before photobleaching is dependent upon the molecular structure and the local environment. Some fluorophores bleach quickly after emitting only a few photons, while others that are more robust can undergo thousands or millions of cycles before bleaching. This interactive tutorial explores variations in photobleaching rates in single, dual, and multiply labeled fluorescence specimens.

Reference Listing - 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. Green Fluorescence Protein is rapidly becoming a labeling method of choice for molecular and cellular biologists who can now explore biochemical events in living cells with natural fluorophores. Taken together, these and other important advances have propelled the visualization of living cells tagged with specific fluorescent probes into the mainstream of research in a wide spectrum of disciplines. The reference materials listed below were utilized in the construction of the fluorescence section of the Molecular Expressions Microscopy Primer.