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Fluorescence Microscopy

Introduction to Fluorescence

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.

Pioneers in Fluorescence

Alexandre Edmond Becquerel (1820-1891) - During his investigations into the nature of fluorescence and phosphorescence, Becquerel invented the phosphoroscope, a device capable of measuring the duration of time between the exposure of a solid, liquid, or gas to a light source and the substance's exhibition of phosphorescence. Through the use of the phosphoroscope, the physicist was able to more accurately determine whether or not certain materials exhibited phosphorescence or fluorescence. The phosphoroscope also enabled Becquerel to discover phosphorescence in a number of materials that were previously not believed to exhibit the effect.

Alexander Jablonski (1898-1980) - Born in the Ukraine in 1898, Alexander Jablonski is best known as the father of fluorescence spectroscopy. Jablonski's primary scientific interest was the polarization of photoluminescence in solutions, and in order to explain experimental evidence gained in the field, he differentiated the transition moments between absorption and emission. His work resulted in his introduction of what is now known as a Jablonski Energy Diagram, a tool that can be used to explain the kinetics and spectra of fluorescence, phosphorescence, and delayed fluorescence.

Johan Sebastiaan Ploem (1927-Present) - Johan Ploem, a renowned scientist, has been a physician, educator and researcher, but is most famous for his invention of the epi-illumination cube used in fluorescence microscopy. Ploem's vertical illuminator bears his name and is commonly used today. The design consists of an excitation filter, dichroic mirror (or beamsplitter), and a barrier (or emission) filter housed together in a small cube. In addition to solving lighting problems previously incurred in fluorescence microscopy, Ploem's illumination cube has made it a simple process to change fluorescence filter combinations by rotating a knob or translating a lever.

George Gabriel Stokes (1819-1903) - Throughout his career, George Stokes emphasized the importance of experimentation and problem solving, rather than focusing solely on pure mathematics. His practical approach served him well and he made important advances in several fields, most notably hydrodynamics and optics. Stokes coined the term fluorescence, discovered that fluorescence can be induced in certain substances by stimulation with ultraviolet light, and formulated Stokes Law in 1852. Sometimes referred to as Stokes shift, the law holds that the wavelength of fluorescent light is always greater than the wavelength of the exciting light. An advocate of the wave theory of light, Stokes was one of the prominent nineteenth century scientists that believed in the concept of an ether permeating space, which he supposed was necessary for light waves to travel.

Gregorio Weber (1916-1997) - At Cambridge University in England, Gregorio Weber's thesis advisor suggested he study the fluorescence of flavins and flavoproteins, instigating the beginning of a long, successful career that resulted in Weber becoming generally recognized as the founder of modern fluorescence spectroscopy. Among the many groundbreaking feats that Weber achieved in the field of fluorescence was the introduction of fluorescence polarization as a method to study macromolecular dynamics, the creation of the first broadly utilized phase-modulation fluorometer, and the presentation of the first report regarding the classical technique of measuring the absolute quantum yield of fluorescence.

Interactive Java Tutorials

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.

Selected References

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.

Contributing Authors

Brian Herman and Victoria E. Centonze Frohlich - Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229.

Joseph R. Lakowicz - Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland and University of Maryland Biotechnology Institute (UMBI), 725 West Lombard Street, Baltimore, Maryland 21201.

Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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