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.
Fundamental Aspects of Fluorescence Microscopy - The modern fluorescence microscope combines the power of high performance optical components with computerized control of the instrument and digital image acquisition to achieve a level of sophistication that far exceeds that of simple observation by the human eye. Microscopy now depends heavily on electronic imaging to rapidly acquire information at low light levels or at visually undetectable wavelengths, as discussed in this Nikon MicroscopyU review article. These technical improvements are not mere window dressing, but are essential components of the light microscope as a system.
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.
Overview of Light Sources for Fluorescence Microscopy - 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.
Light Sources for Optical Microscopy - The performance of the various illumination sources available for optical microscopy depends on the emission characteristics and geometry of the source, as well as the focal length, magnification and numerical aperture of the collector lens system. In gauging the suitability of a particular light source, the important parameters are structure (the spatial distribution of light, source geometry, coherence, and alignment), the wavelength distribution, spatial and temporal stability, brightness, and to what degree these various parameters can be controlled.
Focusing and Alignment of Arc Lamps - Mercury and xenon arc lamps are now widely utilized as illumination sources for a large number of investigations in widefield fluorescence microscopy. Visitors can gain practice aligning and focusing the arc lamp in a Mercury or Xenon Burner with this Nikon MicroscopyU interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope.
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.
Introduction to Fluorescent Proteins - The discovery of green fluorescent protein in the early 1960s ultimately heralded a new era in cell biology by enabling investigators to apply molecular cloning methods, fusing the fluorophore moiety to a wide variety of protein and enzyme targets, in order to monitor cellular processes in living systems using optical microscopy and related methodology. When coupled to recent technical advances in widefield fluorescence and confocal microscopy, including ultrafast low light level digital cameras and multitracking laser control systems, the green fluorescent protein and its color-shifted genetic derivatives have demonstrated invaluable service in many thousands of live-cell imaging experiments.
Choosing Fluorophore Combinations for Confocal Microscopy - In planning multiple label fluorescence staining protocols for widefield and laser scanning confocal fluorescence microscopy experiments, the judicious choice of probes is paramount in obtaining the best target signal while simultaneously minimizing bleed-through artifacts. This interactive tutorial is designed to explore the matching of dual fluorophores with efficient laser excitation lines, calculation of emission spectral overlap values, and determination of the approximate bleed-through level that can be expected as a function of the detection window wavelength profiles.
Specimen Preparation Using Synthetic Fluorophores and Indirect Immunofluorescence - Confocal microscopy was becoming more than just a novelty in the early 1980s due to the upswing in applications of widefield fluorescence to investigate cellular architecture and function. As immunofluorescence techniques, as well as the staining of subcellular structures using synthetic fluorophores, became widely practiced in the late 1970s, microscopists grew increasingly frustrated with their inability to distinguish or record fine detail in widefield instruments due to interference by fluorescence emission occurring above and below the focal plane. Today, confocal microscopy, when coupled to the application of new advanced synthetic fluorophores, fluorescent proteins, and immunofluorescence reagents, is one of the most sophisticated methods available to probe sub-cellular structure. The protocols described in this section address the specimen preparation techniques using synthetic fluorophores coupled to immunofluorescence that are necessary to investigate fixed adherent cells and tissue cryosections using widefield and confocal fluorescence microscopy.
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.
Advanced Techniques in Fluorescence Microscopy
NEW! - Laser Scanning Confocal Microscope Simulator - Perhaps the most significant advance in optical microscopy during the past decade has been the refinement of mainstream laser scanning confocal microscope (LSCM) techniques using improved synthetic fluorescent probes and genetically engineered proteins, a wider spectrum of laser light sources coupled to highly accurate acousto-optic tunable filter control, and the combination of more advanced software packages with modern high-performance computers. This interactive tutorial explores multi-laser fluorescence and differential interference contrast (DIC) confocal imaging using the Olympus FluoView FV1000 confocal microscope software interface as a model.
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.
Live-Cell Imaging - An increasing number of investigations are using live-cell imaging techniques to provide critical insight into the fundamental nature of cellular and tissue function, especially due to the rapid advances that are currently being witnessed in fluorescent protein and synthetic fluorophore technology. As such, live-cell imaging has become a requisite analytical tool in most cell biology laboratories, as well as a routine methodology that is practiced in the wide ranging fields of neurobiology, developmental biology, pharmacology, and many of the other related biomedical research disciplines. Among the most significant technical challenges for performing successful live-cell imaging experiments is to maintain the cells in a healthy state and functioning normally on the microscope stage while being illuminated in the presence of synthetic fluorophores and/or fluorescent proteins.
Olympus FluoView Confocal Microscopy Resource Center - The new Olympus FluoViewTM 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.
Spinning Disk Confocal Microscopy - Spinning disk microscopy has advanced significantly in the past decade and now represents one of the optimum solutions for both routine and high-performance live-cell imaging applications. The rapid expansion in biomedical research using live-cell imaging techniques over the past several years has been fueled by a combination of events that include dramatic advances in spinning disk confocal microscopy instrumentation coupled with the introduction of novel ultra-sensitive detectors and continued improvements in the performance of genetically-encoded fluorescent proteins.
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.
Spectral Imaging and Linear Unmixing - Spectral imaging and linear unmixing is becoming an important staple in the microscopist's toolbox, particularly when applied to the elimination of autofluorescence and for FRET investigations. Instruments equipped for spectral imaging are becoming increasingly popular and many confocal microscopes now offer this capability. Widefield fluorescence and brightfield microscopy are also being used more frequently for resolving complex fluorophore and absorbing dye mixtures, a trend that should continue into the future.
Fluorescence in situ Hybridization: Hardware and Software Implications in the Research Laboratory - The power of in situ hybridization can be greatly extended by the simultaneous use of multiple fluorescent colors. Multicolor fluorescence in situ hybridization (FISH), in its simplest form, can be used to identify as many labeled features as there are different fluorophores used in the hybridization. By using not only single colors, but also combinations of colors, many more labeled features can be simultaneously detected in individual cells using digital imaging microscopy.
Epi-Fluorescence Illumination for Stereomicroscopy - The application of stereomicroscopes for GFP observation is now so prevalent that stereo fluorescence illuminators are more frequently referred to as GFP illuminators, even though they can be utilized for many other applications in both the life sciences and the electronics manufacturing industry. Large specimens, such as larvae, nematodes, Zebrafish, oocytes, and mature insects can be easily selected and manipulated when they are labeled with GFP and illuminated by fluorescence techniques.
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 Galleries
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 Nikon DXM 1200 digital camera, an Optronics MagnaFire Peltier-cooled camera, 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.
Nikon Fluorescence Microscopy Digital Image Gallery - The widefield reflected light fluorescence microscope has been a fundamental tool for the examination of fluorescently labeled cells and tissues since the introduction of the dichromatic mirror in the late 1940s. Furthermore, advances in synthetic fluorophore design coupled to the vast array of commercially available primary and secondary antibodies have provided the biologist with a powerful arsenal in which to probe the minute structural details of living organisms with this technique. In the late twentieth century, the discovery and directed mutagenesis of fluorescent proteins added to the cadre of tools and created an avenue for scientists to probe the dynamics of living cells in culture. This gallery examines the fluorescence microscopy of both cells and tissues with a wide spectrum of fluorescent probes.
Anatomy of the Fluorescence Microscope
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.
Interactive Java Tutorials
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.
References and Literature Sources
General Fluorescence Microscopy Literature Sources - 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.
ZEISS Campus Fluorescence Microscopy Reference Library - The introduction of genetically-encoded fluorescent protein fusions as a localization marker in living cells has revolutionized the field of cell biology, and the application of photostable quantum dots looms on the horizon. Live-cell imaging techniques now involved a wide spectrum of imaging modalities, including widefield fluorescence, confocal, multiphoton, total internal reflection, FRET, lifetime imaging, superresolution, and transmitted light microscopy. The references listed in this section point to review articles that should provide the starting point for a thorough understanding of live-cell imaging.
Fluorescent Protein Literature Sources - The disciplines of cellular and molecular biology are being rapidly and dramatically transformed by the application of fluorescent proteins developed from marine organisms as fusion tags to track protein behavior in living cells. The most widely used of these probes, green fluorescent protein, can be attached to virtually any target of interest and still fold into a viable fluorescent species. The resulting chimera can be employed to localize previously uncharacterized proteins or to visualize and track known proteins to further understand critical events at the cellular and molecular levels. This section features a bibliography of literature sources for review articles and original research reports on the discovery, applications, and continued development of fluorescent proteins.
Daniel Axelrod - Department of Biophysics, 930 North University Ave., University of Michigan, Ann Arbor, Michigan 48109.
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.
David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, 702 Light Hall, Nashville, Tennessee, 37212.
Christopher Hardee, Roy Kinoshita, Travis Wakefield, and Robert Johnson - Omega Optical, Inc., 210 Main Street, Brattleboro, Vermont, 05301.
Turan Erdogan - Semrock, Inc., 3625 Buffalo Road, Rochester, New York, 14624.
Mortimer Abramowitz, William K. Fester, Yoshihiro Kawano, and Reinhard G. Enders - Olympus America, Inc., Two Corporate Center Drive, Melville, New York, 11747.
Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.
Matthew J. Parry-Hill, Thomas J. Fellers, Nathan S. Claxton 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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