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 asfading) 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.