The Fluorescence Microscope
In fluorescence microscopy, wide variations between localized fluorophore concentrations within the specimen, coupled to differences in extinction coefficient and quantum yield from one fluorochrome to another, significantly influence the emission signal produced for a given quantity of excitation intensity. Considering that many specimens contain only minute quantities of fluorescent material in any particular viewfield, these combined factors produce an average level of fluorescence emission that is four to six orders of magnitude less than the excitation intensity. In addition, several of the more sophisticated fluorescence techniques, such as in situ hybridization and resonance energy transfer (FRET), have emission signal intensities that can range nine to ten orders of magnitude less than that of the excitation. In order to compensate for these large discrepancies between the intensity of excitation and emission, modern fluorescence microscopes must be able to attenuate the excitation illumination by levels exceeding a billion times without disturbing the fluorescence signal.
Anatomy of the Fluorescence Microscope - Fluorescence microscopes have evolved with amazing speed over the past decade, coupled to equally rapid advances in laser technology, solid-state detectors, interference thin film fabrication, and computer-based image analysis. The development of high numerical aperture water immersion objectives has further assisted investigations of biological phenomena, allowing investigators to probe deep within the living cell in its natural environment. As the microscope manufacturers respond to the changing needs of the research community, the development of advanced fluorescence instruments and accessories will no doubt continue, and their ultimate contribution to explorations into nature's mysteries may ultimately be of profound significance.
Fluorescence Microscopy with Transmitted Light - Transmitted (or Diascopic) illumination was once the primary method of illumination for fluorescence samples. Unfortunately, in brightfield transmitted light fluorescence, it is difficult to separate the excitation light from the fluorescing light because both kinds of light directly enter the objective. This section discusses various aspects of transmitted fluorescence illumination and the equipment configurations necessary to perform this type of microscopy.
Fluorescence Microscope Schematic Diagrams
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.
Reflected Light Fluorescence Microscopy Light Pathways - In reflected light Köhler illumination for fluorescence microscopy, an image of the light source is focused by the collector lens onto the aperture iris diaphragm located in the vertical illuminator. This diaphragm shares a conjugate plane with the rear aperture of the objective and the lamp arc or filament, and therefore, determines the illuminated field aperture size. Together, the light source, vertical illuminator aperture diaphragm, and objective rear focal plane (pupil) form the illumination set of conjugate planes. Opening or closing the aperture diaphragm is used to control stray light and regulate the intensity (numerical aperture) of illumination without altering the size of the illuminated field. In the image, adjustment of the aperture diaphragm affects brightness and contrast. This interactive tutorial explores the light pathways in a reflected light (episcopic) fluorescence microscope.
Upright Epi-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 Epi-Fluorescence 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.
Mortimer Abramowitz and William K. Fester - Olympus America, Inc., Two Corporate Center Drive., Melville, New York, 11747.
Brian Herman - Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229.
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.
Matthew Parry-Hill 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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