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.
Introduction to Fluorescence - 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. The use of fluorochromes has made it possible to identify cells and sub-microscopic cellular components and other entities with a high degree of specificity amidst non-fluorescing material. What is more, the fluorescence microscope can reveal the presence of fluorescing material with exquisite sensitivity. An extremely small number of fluorescent molecules (as few as 50 molecules per cubic micrometer) can be detected. In a given sample, through the use of multiple staining, different probes will reveal the presence of individual target molecules. Although the fluorescence microscope cannot provide spatial resolution below the diffraction limit of the respective specimens, the presence of fluorescing molecules below such limits is made remarkably visible.
Overview of Excitation and Emission Fundamentals - When electrons go from the excited state to the ground state, there is a loss of vibrational energy. As a result, the emission spectrum is shifted to longer wavelengths than the excitation spectrum (wavelength varies inversely to radiation energy). This phenomenon is known as Stokes Law or Stokes shift. The greater the Stokes shift, the easier it is to separate excitation light from emission light. The emission intensity peak is usually lower than the excitation peak; and the emission curve is often a mirror image of the excitation curve, but shifted to longer wavelengths. To achieve maximum fluorescence intensity, the fluorochrome is usually excited at the wavelength at the peak of the excitation curve, and the emission is selected at the peak wavelength (or other wavelengths chosen by the observer) of the emission curve. The selections of excitation wavelengths and emission wavelengths are controlled by appropriate filters. In determining the spectral response of an optical system, technical corrections are required to take into account such factors as glass transmission and detector sensitivity variables for different wavelengths.
John Frederick William Herschel (1792-1871) - John Herschel was the only child of renowned scientist and astronomer William Herschel. In 1820, the younger Herschel was one of the founding members of the Royal Astronomical Society, and when his father died in 1822 he carried on with the elder Herschel's work, making a detailed study of double stars. In collaboration with James South Herschel compiled a catalog of observations that was published in 1824. The work garnered the pair the Gold Medal from the Royal Astronomical Society and the Lalande Prize from the Paris Academy of Sciences. In 1839, Herschel developed a technique for creating photographs on sensitized paper, independently of William Fox Talbot, but did not attempt to commercialize the process. However, he published several papers on photographic processes and was the first to utilize the terms positive and negative in reference to photography. Particularly important to the future of science, in 1845 Herschel reported the first observation of the fluorescence of a quinine solution in sunlight.
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.
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.
Interactive Java Tutorials
Electron Excitation and Emission - Electrons can absorb energy from external sources, such as lasers, arc-discharge lamps, and tungsten-halogen bulbs, and be promoted to higher energy levels. This tutorial explores how photon energy is absorbed by an electron to elevate it into a higher energy level and how the energy can subsequently be released, in the form of a lower energy photon, when the electron falls back to the original ground state.
Fluorescence Filter Spectral Transmission Profiles - Fluorescence microscopes are equipped with a combination of three essential filters (often termed a filter set) that are positioned in the optical pathway between the light source in the vertical illuminator and the objective. The filters are strategically oriented within a specialized cube or block that enables the illumination to enter from one side and pass to and from the specimen in defined directions along the microscope optical axis. This tutorial explores the spectral overlap regions of fluorescence filter combinations, and how changes to the individual filter properties help determine the bandwidth of wavelengths passed through the various filter sets.
Jablonski Energy Diagram - Fluorescence activity can be schematically illustrated with the classical Jablonski diagram, first proposed by Professor Alexander Jablonski in 1935 to describe absorption and emission of light. Prior to excitation, the electronic configuration of the molecule is described as being in the ground state. Upon absorbing a photon of excitation light, usually of short wavelengths, electrons may be raised to a higher energy and vibrational excited state, a process that may only take a quadrillionth of a second (a time period commonly referred to as a femtosecond, 10E-15 seconds). This tutorial explores 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.
Selected Literature References
Reference Listing - The field of fluorescence spectroscopy and microscopy is experiencing a renaissance with the introduction of new techniques such as confocal, multiphoton, deconvolution, time-resolved investigations, and total internal reflection, coupled to the current 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 introductory fluorescence section in the Molecular Expressions Microscopy Primer.
Mortimer Abramowitz - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Ian D. Johnson, Matthew J. Parry-Hill, Brian O. Flynn, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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