In the rapidly expanding fields of cellular and molecular biology, widefield and confocal fluorescence illumination and observation is becoming one of the techniques of choice. These techniques, which are almost universally employed in both the medical and biological sciences, have spurred the development of more sophisticated microscopes and numerous fluorescence accessories.

Introduction to 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. These technical improvements are not mere window dressing, but are essential components of the light microscope as a system.

Nikon Fluorescence Filter Sets - Epi-Fluorescence interference filter combinations are housed in a filter cube and include an excitation filter, dichroic mirror (or beamsplitter), and a barrier (or emission) filter. Use this guide in selecting the appropriate filter set to match the spectral excitation and emission characteristics of chromophores used in fluorescence microscopy experiments.

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.

Imaging Parameters for Fluorescent Proteins - The wide spectrum of fluorescent proteins and derivatives uncovered thus far are quite versatile and have been successfully employed in almost every biological discipline from microbiology to systems physiology. These unique probes have proven extremely useful as reporters for gene expression studies in both cultured cells and entire animals. In living cells, fluorescent proteins are most commonly utilized to track the localization and dynamics of proteins, organelles, and other cellular compartments, as well as a tracer of intracellular protein trafficking. Quantitative imaging of fluorescent proteins is readily accomplished with a variety of techniques, including widefield, confocal, and multiphoton microscopy, to provide a unique window for exposing the intricacies of cellular structure and function.

TIRF Microscopy: Introduction and Applications - Various mechanisms are often employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. Total internal reflection fluorescence microscopy (TIRFM) exploits the unique properties of an induced evanescent wave or field in a limited specimen region immediately adjacent to the interface between two media having different refractive indices. In practice, the most commonly utilized interface in the application of TIRFM is the contact area between a specimen and a glass coverslip or tissue culture container.

Fundamentals and Applications in Multiphoton Microscopy - Two-photon excitation microscopy offers great utility for dynamic imaging of living cells in thick specimens, such as intact tissue. The technique makes possible many experiments in which conventional imaging cannot be performed, or would not provide the information desired. Relying on mode-locked (pulsed) laser illumination to produce sufficient photon density at the focal point, two-photon excitation occurs only in the focal plane. The benefit of localized excitation is that emission is restricted to the narrow focal region, providing sectioning ability without the use of a pinhole. Furthermore, the limited excitation region reduces phototoxicity because photodamage is largely confined to the focal volume.

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.

Fluorescence Illumination in 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. The fluorescence illumination reveals which organisms are producing the fluorescent protein and the stereoscopic vision coupled to a large field of view and ample working distance enables observers to conduct experiments with forceps, pipettes, or micromanipulators. Other, more conventional, specimens are also easily observed and recorded using stereomicroscopes with fluorescence illumination.

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.

Laser Safety - The two major concerns in safe laser operation are exposure to the beam and the electrical hazards associated with high voltages within the laser and its power supply. While there are no known cases of a laser beam contributing to a person's death, there have been several instances of deaths attributable to contact with high voltage laser-related components. Beams of sufficiently high power can burn the skin, or in some cases create a hazard by burning or damaging other materials, but the primary concern with regard to the laser beam is potential damage to the eyes, which are the part of the body most sensitive to light.

Focus and Alignment of Mercury and Xenon 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 interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope.

Fluorescence Filter Noise Terminator - An innovative new fluorescence filter block design by Nikon helps to eliminate the possibility of residual stray light that occurs in the microscope fluorescence optical pathway, vastly improving the emission signal-to-noise ratio. Termed the Noise Terminator, this technology directs deviated stray light away from the objective light collection path, resulting in a dramatic improvement in image contrast. This interactive tutorial demonstrates how the Noise Terminator technology functions.

Matching Fluorescent Probes with Fluorescence Filter Blocks - Modern fluorescence microscope instrumentation utilizes a combination of filters in conjunction with a dichromatic beam splitter to satisfy the excitation and emission requirements of the fluorescent probe(s) used to label the specimen. When these components are chosen appropriately, the microscope provides an essential mechanism for selective excitation of specimen fluorophores, and the subsequent isolation of much weaker fluorescence emission necessary for image formation. By carefully matching excitation and emission filter properties with the function of the dichromatic beamsplitter, labeled specimen features are imaged on a dark background with maximum sensitivity. This interactive tutorial enables visitors to determine optimum choices among current Nikon fluorescence filter blocks for maximizing the efficiency of excitation and emission with specific fluorescent probes.

Choosing Filter Combinations for Fluorescent Proteins - Fluorescence filter combinations designed to image fluorescent proteins must be carefully chosen to maximize the level of emission intensity presented to the detector while simultaneously reducing the number of unwanted photons from autofluorescence or bleed-through by other fluorophores. The broad absorption and emission spectral profiles exhibited by most fluorescent proteins offer a wide range of choice in filters, which are usually optimized for use with a specific detection system (human eye, digital camera, or photomultiplier). This interactive tutorial is designed to enable the identification of critical filter parameters, including the center wavelength, bandwidth region, and dichromatic mirror cut-on wavelength, which are necessary for imaging fluorescent proteins.

Choosing Fluorescent Proteins for Dual Labeling Experiments - The broad excitation and emission spectral profiles exhibited by fluorescent proteins and their color-shifted genetic variants often require specialized considerations when designing live-cell imaging experiments using two or more of these unique probes simultaneously. Of primary concern are potential bleed-through artifacts resulting from the significant degree of emission spectral overlap usually exhibited by fluorescent protein combinations. This interactive tutorial explores matching fluorescent proteins for dual labeling investigations with regards to spectral bandwidth and overlap, excitation efficiency, emission window dimensions, and other parameters necessary to design logical experiments.

Balancing Arc-Discharge Lamp Excitation Illumination - Fine-tuning of the fluorescence microscope excitation spectrum for imaging dual or multiply labeled specimens can be readily accomplished with a split-filter excitation balancer, which contains tandem shortpass and longpass interference filters that are translated across the illumination aperture to adjust the arc-discharge lamp wavelength distribution profile. This interactive tutorial explores how the Nikon Eclipse i-Series excitation balancer system affects the fluorescence emission intensity of multiply labeled specimens when employed in conjunction with dual and triple excitation band filter combinations.

Stereomicroscopy Fluorescence - The illuminator for epi-fluorescence on a stereomicroscope functions in a manner that is similar to those employed on more complex compound microscopes. Typically, the fluorescence illuminator consists of a xenon or mercury arc lamp contained in an external lamphouse that is attached to the microscope via an intermediate tube (or vertical illuminator) positioned between the microscope zoom body and observation tubes. This interactive tutorial explores internal optical pathways of the Nikon SMZ1500 stereomicroscope equipped with an epi-illumination intermediate tube and lamphouse.

Widefield 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.

Stereomicroscopy Fluorescence Image Gallery - 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 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 techiques. This technique is also applicable to traditional fluorescence specimens, such as stained thin sections, cell culture mounts, and autofluorescence in plant tissues. Visit the gallery to observe the wide variety of specimens imaged using this novel new technique.