Fluorescence illumination and observation is the most rapidly expanding microscopy technique employed today, both in the medical and biological sciences, a fact which has spurred the development of more sophisticated microscopes and numerous fluorescence accessories. Epi-fluorescence, or incident light fluorescence, has now become the method of choice in many applications and comprises a large part of this tutorial. We have divided the fluorescence section of the primer into several categories to make it easier to organize and download. Please follow the links below to navigate to points of interest.

Introductory Concepts - 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.

Anatomy of the Fluorescence Microscope - In contrast to other modes of optical microscopy that are based on macroscopic specimen features, such as phase gradients, light absorption, and birefringence, fluorescence microscopy is capable of imaging the distribution of a single molecular species based solely on the properties of fluorescence emission. Thus, using fluorescence microscopy, the precise location of intracellular components labeled with specific fluorophores can be monitored, as well as their associated diffusion coefficients, transport characteristics, and interactions with other biomolecules. In addition, the dramatic response in fluorescence to localized environmental variables enables the investigation of pH, viscosity, refractive index, ionic concentrations, membrane potential, and solvent polarity in living cells and tissues.

Practical Aspects of Fluorescence Filter Combinations - Microscope manufacturers provide proprietary filter combinations (often referred to as cubes or blocks) that contain a combination of dichroic mirrors and filters capable of exciting fluorescent chromophores and diverting the resulting secondary fluorescence to the eyepieces or camera tube. A wide spectrum of filter cubes is available from most major manufacturers, which now produce filter sets capable of imaging most of the common fluorophores in use today.

Light Sources - In order to generate enough excitation light intensity to furnish secondary fluorescence emission capable of detection, powerful light sources are needed. These are usually either mercury or xenon arc (burner) lamps, which produce high-intensity illumination powerful enough to image faintly visible fluorescence specimens.

Optimization and Troubleshooting - A key feature of fluorescence microscopy is its ability to detect fluorescent objects that are sometimes faintly visible or even very bright relative to the dark (often black) background. In order to optimize this feature, image brightness and resolution must be maximized using the principles discussed in this section. We also review common problems with microscope configuration in fluorescence microscopy.

Electronic Imaging Detectors - The range of light detection methods and the wide variety of imaging devices currently available to the microscopist make the selection process difficult and often confusing. This discussion is intended to aid in understanding the basics of light detection and to provide a guide for selecting a suitable detector for specific applications in fluorescence microscopy.

Introduction to Fluorophores - Widefield fluorescence and laser scanning confocal microscopy rely heavily on secondary fluorescence emission as an imaging mode, primarily due to the high degree of sensitivity afforded by the techniques coupled with the ability to specifically target structural components and dynamic processes in chemically fixed as well as living cells and tissues. Many fluorescent probes are constructed around synthetic aromatic organic chemicals designed to bind with a biological macromolecule. Fluorescent dyes are also useful in monitoring cellular integrity (live versus dead and apoptosis), endocytosis, exocytosis, membrane fluidity, protein trafficking, signal transduction, and enzymatic activity. In addition, fluorescent probes have been widely applied to genetic mapping and chromosome analysis in the field of molecular genetics.

Optical Highlighter Fluorescent Proteins - Protein chromophores that can be activated to initiate fluorescence emission from a quiescent state (a process known as photoactivation), or are capable of being optically converted from one fluorescence emission bandwidth to another (photoconversion), represent perhaps the most promising approach to the in vivo investigation of protein lifetimes, transport, and turnover rates. Appropriately termed molecular or optical highlighters, photoactivated fluorescent proteins generally display little or no initial fluorescence under excitation at the imaging wavelength, but dramatically increase their fluorescence intensity after activation by irradiation at a different (usually lower) wavelength. Photoconversion optical highlighters, on the other hand, undergo a change in the fluorescence emission bandwidth profile upon optically-induced changes to the chromophore. These effects result in the direct and controlled highlighting of distinct molecular pools within the cell.

Fluorescence Photomicrography - Photomicrography under fluorescence illumination conditions presents a unique set of circumstances posing special problems for the microscopist. Exposure times are often exceedingly long, the specimen's fluorescence may fade during exposure, and totally black backgrounds often inadvertently signal light meters to suggest overexposure.

Glossary of Terms in Fluorescence and Confocal Microscopy - The complex nomenclature of fluorescence microscopy is often confusing to both beginning students and seasoned research microscopists alike. This resource is provided as a guide and reference tool for visitors who are exploring the large spectrum of specialized topics in fluorescence and laser scanning confocal microscopy.

Introduction to Confocal Microscopy - Confocal microscopy offers several advantages over conventional optical microscopy, including controllable depth of field, the elimination of image degrading out-of-focus information, and the ability to collect serial optical sections from thick specimens. The key to the confocal approach is the use of spatial filtering to eliminate out-of-focus light or flare in specimens that are thicker than the plane of focus. There has been a tremendous explosion in the popularity of confocal microscopy in recent years, due in part to the relative ease with which extremely high-quality images can be obtained from specimens prepared for conventional optical microscopy, and in its great number of applications in many areas of current research interest.

Olympus FluoView Laser Scanning Confocal Microscopy - The new Olympus FluoView^TM FV1000 is the latest in point-scanning, point-detection, confocal laser scanning microscopes designed for today's intensive and demanding biological research investigations. Excellent resolution, bright and crisp optics, and high efficiency of excitation, coupled to an intuitive user interface and affordability are key characteristics of this state-of-the-art optical microscopy system.

Multiphoton Excitation Microscopy - Multiphoton fluorescence microscopy is a powerful research tool that combines the advanced optical techniques of laser scanning microscopy with long wavelength multiphoton fluorescence excitation to capture high-resolution, three-dimensional images of specimens tagged with highly specific fluorophores.

Fluorescence Resonance Energy Transfer (FRET) - The precise location and nature of the interactions between specific molecular species in living cells is of major interest in many areas of biological research, but investigations are often hampered by the limited resolution of the instruments employed to examine these phenomena. Conventional widefield fluorescence microscopy enables localization of fluorescently labeled molecules within the optical spatial resolution limits defined by the Rayleigh criterion, approximately 200 nanometers (0.2 micrometer). However, in order to understand the physical interactions between protein partners involved in a typical biomolecular process, the relative proximity of the molecules must be determined more precisely than diffraction-limited traditional optical imaging methods permit. The technique of fluorescence resonance energy transfer (more commonly referred to by the acronym FRET), when applied to optical microscopy, permits determination of the approach between two molecules within several nanometers, a distance sufficiently close for molecular interactions to occur.

Total Internal Reflection Fluorescence Microscopy - Total internal reflection fluorescence microscopy (TIRFM) is an elegant optical technique utilized to observe single molecule fluorescence at surfaces and interfaces. The technique is commonly employed to investigate the interaction of molecules with surfaces, an area which is of fundamental importance to a wide spectrum of disciplines in cell and molecular biology.

Laser Systems for Optical Microscopy - The lasers commonly employed in optical microscopy are high-intensity monochromatic light sources, which are useful as tools for a variety of techniques including optical trapping, lifetime imaging studies, photobleaching recovery, and total internal reflection fluorescence. In addition, lasers are also the most common light source for scanning confocal fluorescence microscopy, and have been utilized, although less frequently, in conventional widefield fluorescence investigations.

Fluorescence and Phase Contrast Combination Microscopy - To minimize the effects of photobleaching, fluorescence microscopy can be combined with phase contrast illumination. The idea is to locate the specific area of interest in a specimen using the non-destructive contrast enhancing technique (phase) then, without relocating the specimen, switch the microscope to fluorescence mode.

Fluorescence and Differential Interference Contrast Combination Microscopy - Fluorescence microscopy can also be combined with contrast enhancing techniques such as differential interference contrast (DIC) illumination to minimize the effects of photobleaching by locating a specific area of interest in a specimen using DIC then, without relocating the specimen, switching the microscope to fluorescence mode.

Fluorescence Microscopy Digital Image Gallery - Featuring specimens collected from a wide spectrum of disciplines, the fluorescence gallery contains a variety of examples using both specific fluorochrome stains and autofluorescence. Images were captured utilizing either a digital camera systems or classical photomicrography on film with Fujichrome Provia 35 millimeter transparency film.

Fluorescence Microscopy of Cells in Culture - Serious attempts at the culture of whole tissues and isolated cells were first undertaken in the early 1900s as a technique for investigating the behavior of animal cells in an isolated and highly controlled environment. The term tissue culture arose because most of the early cells were derived from primary tissue explants, a technique that dominated the field for over 50 years. As established cell lines emerged, the application of well-defined normal and transformed cells in biomedical investigations has become an important staple in the development of cellular and molecular biology. This fluorescence image gallery explores over 30 of the most common cell lines, labeled with a variety of fluorophores using both traditional staining methods as well as immunofluorescence techniques.

Olympus BX51 Upright Microscope - The modern upright epi-fluorescence microscope is equipped with a vertical illuminator that contains a turret of filter cubes and a mercury or xenon arc lamp housing. Light passes from the lamphouse thorough field and aperture diaphragms and into a cube that contains both excitation and emission filters and a dichroic mirror. After passing through the objective and being focused onto the specimen, reflected excitation and secondary fluorescence are filtered upon return through the cube. Next, the light (primarily secondary fluorescence) is routed to the eyepieces or detector.

Olympus IX70 Inverted Microscope - Microscopes with an inverted-style frame are designed primarily for tissue culture applications and are capable of producing fluorescence illumination through an episcopic and optical pathway. Epi-illuminators usually consist of a mercury or xenon lamphouse (or laser system) stationed in a port at the rear of the microscope frame. Fluorescence illumination from the arc lamp passes through a collector lens and into a cube that contains a set of interference filters, including a dichroic mirror, barrier filter, and excitation filter. After excitation of the specimen, secondary fluorescence is collected by the objective and directed through the microscope optical train.

Fluorescence Microscope Light Pathways - This interactive tutorial explores illumination pathways in the Olympus BX51 research-level upright microscope. The microscope drawing presented in the tutorial illustrates a cut-away diagram of the Olympus BX51 microscope equipped with a vertical illuminator and lamphouses for both diascopic (tungsten-halogen) and epi-fluorescence (mercury arc) light sources. Sliders control illumination intensity and enable the visitor to select from a library of five fluorescence interference filter combinations that have excitation values ranging from the near ultraviolet to long-wavelength visible light.

Inverted Microscope Light Pathways - Explore light pathways through an inverted tissue culture microscope equipped with for both diascopic (tungsten-halogen) and epi-fluorescence (mercury arc) illumination. Light intensity through the pathways the in microscope are controllable with sliders, as is a library of five fluorescence interference filter combinations that have excitation values ranging from the near ultraviolet to long-wavelength visible light. The "virtual" inverted microscope is also equipped with traditional (35 millimeter) and CCD camera systems to enable the visitor to observe how light rays are directed into these peripheral devices.

Selected Literature References - 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.