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Glossary

Aberration, Chromatic: A characteristic in a lens or optical system due to the greater refraction of shorter wavelengths over that of longer ones at a lens surface. Hence the focal length of a simple lens is shorter for blue than for red rays. This dispersion of the wave-lengths will cause color fringes in the image field of a lens with such an aberration.

Aberration, Spherical: A lens characteristic whereby image forming rays of one color passing through the outer zones of a lens come to focus at a different distance from the lens than do those of more central rays.

Airy Disk: The bright disk of light (surrounded by alternating dark and bright diffraction rings) that is formed by a perfect diffraction limited lens, focusing an image of an infinitely small source of light. For a minute absorbing spot, the diffraction pattern is a dark airy disk surrounded by brighter and darker diffraction rings. Since the airy disk is the smallest unit that makes up the image of a luminous or absorbing object (formed by a properly corrected microscope lens in focus), the radius of the disk determines the limit of resolution of the microscope.

Back Aperture: The exit pupil of a microscope objective lens. The objective lens back aperture, which can be examined with a phase telescope or by inserting a Bertrand lens, displays the conoscopic interference figure and diffraction patterns.

Bertrand Lens: A small, low power lens, usually on a slide for insertion into the drawtube between analyzer and ocular. It is used to observe the back focal plane of the objective so as to examine interference figures or as an aid in achieving interference figures. It is used to image the lamp filament in setting up Kohler illumination as well as for centering dispersion staining stops to the substage aperture diaphram.

Birefringence: The numerical difference in refractive indices for a substance. In a given crystal view, the interference color (retardation) between crossed polars depends on the birefringence and thickness: Retardation (nm) = 1000 x thickness (nm) x birefringence

Condenser (Substage): In microscopy, the lens mounted before the microscope stage, which transmits light to the object. There are two main categories of condensers: (1) bright field and (2) dark field. All microscope condensers must be carefully focused and aligned for best results.

Confocal Microscopy: Minsky (1957) was the first to propose the technique of confocal microscopy used by laser scanning confocal microscopes. The image seen through a microscope includes the in-focus portion and the out-of-focus portion above and below the plane of focus. The smear or blur produced by the out-of-focus planes is a natural consequence of the optics of the microscope. Confocal microscopy removes out-of-focus haze by passing the light through one or more small apertures, leaving only a thin, highly focused plane. The light from this focused plane can be digitized and stored on a computer. A confocal microscope consists of a standard microscope with a number of complex attachments to direct and process the beam of light. Most confocal microscopes use an intense laser light to scan the specimen. This intense light source is needed to compensate for the light loss which occurs as the light passes through small apertures.

Conjugate Planes/Points: Planes (or points) that are in focus relative to each other. In a microscope adjusted for Koehler illumination, there are two sets of conjugate planes: the aperture planes and the field planes. See field planes, Koehler illumination.

Contrast: A measure of the gradation in luminance that provides gray scale (or color) information. Contrast is expressed as the ratio (difference in luminance)/(average luminance) in adjoining areas of the scene. Under optimum conditions, the human eye can detect the presence of 2% contrast.

Correction Collar: An adjustment collar provided on some high-NA microscope objective lenses. Rotation of the collar adjusts the height of certain lens elements in the objective lens to compensate for variations in coverslip thickness or immersion media. At high NAs, even a small deviation of the coverslip thickness (by as little as a few micrometers in some cases), or refractive index of the immersion medium from the designated standard, can introduce significant aberrations.

Curvature of Field: The image plane formed by a single lens is naturally curved. While one part of the field will be in good focus, the rest will need refocusing to be sharp. While the eye may partially correct for this, a camera lens will not, and the final image as photographed will not be in perfect focus over the entire image plane.

Dark Field Illumination: Any method of illumination which illuminates the specimen but does not admit light directly to the objective. It may be by substage (dark field, q.v.) condensers; by stagespot lighting, by special condensers fitted around special objectives for reflected illumination or by the slit ultramicroscope.

Deconvolution: A mathematical method for applying the point-spread function (PSF) to remove and/or reassign the "out-of-focus" blur in an image specimen. The point spread function of the optical system is either determined experimentally or back calculated from the collected images.

Depth of Field: The depth or thickness of the object space that is simultaneously in acceptable focus.

Depth of Focus: The range of distances between a lens and image plane for which the image formed by the lens at a given setting is clearly focused. With a high-NA microscope objective, the depth of field is very shallow, but the depth of focus can be quite deep and reach several millimeters.

Diaphragm: A fixed or adjustable aperture in an optical system. Diaphragms are used to intercept scattered light, to limit field angles, or to limit image-forming bundles or rays.

Differential Interference Contrast (DIC): A mode of contrast generation in microscopy that yields an image with a shadow relief. The relief reflects the gradient of optical path difference. DIC, which is a form of interference microscopy that uses polarizing beam splitters, can be of the Smith or Nomarski type.

Diffraction: The deviation of light from rectilinear propagation is a characteristic of wave phenomena which occurs when a portion of a wave front is obstructed in some way. When various portions of a wave front propagate past some obstacle, and interfere at a later point past the obstacle, the pattern formed is called a diffraction pattern.

Field Diaphragm: The iris diaphragm that is located in front of the collecting lens of the light source. With Koehler illumination, the condenser focuses the image of the field diaphragm onto the image plane.

Field of View: The extent of the visible image field that can be seen.

Fluorophore: A molecule that absorbs radiation of a specific wavelength range increasing the energy state of the molecule and then subsequently emits radiation of longer wavelengths, i.e., lower energy returning the molecule to the ground energy state.

Fluorescence Recovery After Photobleaching (FRAP): A new technique in light microscopy using a pulse from a focused light source, often a laser microbeam to deplete the fluorescence in a local region in a living cell. The subsequent recovery of fluorescence in the irradiated region is measured to establish the mobility of the molecules that carry the fluorescent tag.

GFP: Green fluorescent protein, GFP, is a spontaneously fluorescent protein isolated from coelenterates, such as the Pacific jellyfish, Aequoria victoria. Its role is to transduce, by energy transfer, the blue chemiluminescence of another protein, aequorin, into green fluorescent light. GFPs can be excited and detected in standard fluorescence microscopes. These molecules provide a powerful method for observing gene expression.

Immersion Liquid: Any liquid occupying the space between the object and microscope objective lens. Such a liquid is usually required by objectives of 3-mm focal length or less. For best results (i.e., resolution) the liquid should be used between the condenser and the microscope slide. Immersion objectives for transmitted light are designed for use with either "oil", glycerin, or water, the refractive index of the liquid and the coverslip (if any) being the determining factor. The liquid and the front lens of the objective should ideally coincide in index and in dispersion value.

Index of Refraction: The ratio of the velocity of light in a vacuum to its velocity through a transparent or translucent substance. For optical materials that velocity is always less than that in a vacuum. Thus, for this class of material the index is always greater than 1.

Interference: Two light beams which originate from the same source and are identical spectrally and in polarization are capable of interference (i.e., are coherent) because they are a wave action.
(1) If they are still in phase or one has been retarded by n*lambda they will add completely to each other's brightness. This is called constructive interference.
(2) If one beam has been retarded, with respect to the other, by lambda/2, i.e., from crest to trough, the two waves nullify each other to the limit of the weaker beam, causing darkness if the two are equal; this is destructive interference. There is only partial interference when the retardation of one beam is only intermediate between lambda and lambda/2. Since complete destructive interference occurs strictly for a single wavelength, independently, it results in residual color when white light is involved.

Koehler Illumination: Mode of microscope illumination in which the light source is imaged onto the condenser iris diaphragm and the field diaphragm (in front of the lamp collector lens) is imaged by the condenser onto the plane of focus of the specimen.

Neutral-density Filter: A light absorbing filter whose absorption spectrum is moderately flat. Depending on the type, the absorption curve is flat primarily in the visible spectral range, or may extend to varying degrees beyond the visible range. For video microscopy, this is an important point since the absorbance may or may not extend into the near-infrared region where the sensitivity of many video image pickup devices is very high.

Nipkow Disk: An opaque circular disk perforated with small holes arranged at equal angular separations and in an Archimedes spiral. The holes trace a raster scanning pattern when the disk is spun around its center. The Nipkow disk was used in early experiments on television and more recently in Petran's confocal microscope.

Numerical Aperture (NA): The numerical aperture of a lens system (objective or condenser). It is the sine of one-half the angular aperture times the refractive index of the medium (1.0 for air, 1.515 for Cargille immersion oil, etc.) between objective and specimen. The numerical aperture is a measure of the light gathering capacity of the lens system and determines its resolving power and depth of field.

Optical Sectioning: The use of high NA objective and condenser lenses on a microscope to achieve a shallow depth of field. With a very shallow depth of field, objects above and below focus contribute little to the in-focus image, so that a clean optical section is obtained. See also depth of field.

Optical Scrambler: An optical device for scrambling the image of a non-uniform light source so that it now fills the condenser aperture uniformly without appreciable loss of total luminous flux through the microscope. The scrambler can be a simple loop of a single optical fiber with its ends appropriately polished.

Parfocal Objectives: Objectives which are mounted so that only a small adjustment of the bodytube and stage is necessary to achieve sharp focus after changing from one objective to any of the others. They are mounted in such a way that the back focal plane is in the same position on the optical axis of the microscope for each objective. Objectives used on a rotating nosepiece are usually parfocal.

Phase Contrast: An optical method devised by F. Zernike for converting the focused image of a phase object (one with differences in refractive index or optical path but not in absorbance), which ordinarily is not visible in focus, into an image with good contrast.

Plan Apochromatic Objective Lens: A modern, high-NA microscope objective lens designed with high degrees of corrections for various aberrations. It is corrected for spherical aberration in four wavelengths (dark blue, blue, green, and red), for chromatic aberration in more than these four wavelengths, and for flatness of field. A single Plan Apo objective may contain as many as 11 lens elements.

Point-spread Function: The mathematical representation of the image of a point source. For a diffraction limited optical system operating in the absence of aberrations, the point-spread function is the Airy disk. See also three dimensional diffraction pattern.

Polarization Colors: Interference colors produced by anisotropic substances placed between two polarizing elements and examined by transmitted white light. See Michel-Levy scale of retardation colors.

Polarized Light: Light that is vibrating in one plane (plane-polarized light), light with a rotary vibration (circular polarized light), or light that is vibrating elliptically (elliptically polarized light). Moonlight and skylight are polarized, as is much reflected light; cloud light is polarized under certain conditions. However, naturally polarized light is, on the whole, rather imperfectly polarized.

Polarized Light Microscope: A microscopical polariscope, i.e., a compound microscope which is equipped with two polars and a Bertrand lens; chemists and mineralogists are the principal users.

Polarizer: A first polarizing element inserted before a preparation. When its vibration direction is at right angles to the vibration direction of the analyzer, the field becomes black if no anisotropic specimen is on the stage or when viewing an anisotropic substance in an extinction position or directly down an optic axis of an anisotropic crystal.

Prism (Optical): A transparent body with at least two polished plane faces inclined with respect to each other, from which light is reflected or through which light is refracted. When light is refracted by a prism whose refractive index exceeds that of the surrounding medium, it is deviated or bent toward the thicker part of the prism.

Quarter-wave Plate: A compensator giving a retardation of about 130 nm, and a phase shift of 1/4, thus constituting a device used with a polarizer and analyzer designed to produce circularly polarized light.

Rayleigh Criterion: A criterion chosen by Lord Rayleigh to define the limit of resolution of a diffraction limited optical instrument. It is the condition that arises when the center of one diffraction pattern is superimposed with the first minimum of another diffraction pattern, produced by a point (or line) source equally bright as the first. For a microscope under this condition, a 26.5% dip in brightness appears between the two maxima, giving rise to the sensation (or probability) of twoness.

Refraction: The change in direction of a ray of light in oblique passage from one transparent medium to another of different density, caused by the effect of a change of velocity of the light waves.

Refractive Index (n): The ratio of the velocity of light in a vacuum to the velocity in a medium. Refractive index generally increases with the atomic number of the constituent atoms.

Resolution: The fineness of detail in an object which is revealed by an optical device. Objectively, resolution is specified as the minimum distance between two lines or points in the object that are perceived as separate by the human eye. Subjectively, the images of the two resolved points must fall on two receptors (rods or cones) which are separated by at least one other receptor on the retina of the eye.

Retardation: The actual distance of one of the doubly refracted rays behind the other as they emerge from an anisotropic substance. It depends on the difference in the two refractive indices, n2 - n1, and the thickness.

S/N (Signal-to-noise Ratio): Also sometimes used as an abbreviation for serial number; can be somewhat confusing in the case of electronic equipment.

Stage Micrometer: A graduated scale used as a standard on the stage of a light microscope for calibrating an eyepiece micrometer; also for determining the magnification of a set-up in photomicrography, etc.

Vignetting: An unintentional, shaded loss of the edges of an image or picture by an optical component clipping the peripheral beams can lead to loss of contrast especially in video microscopy.

Wavelength: In wave motion, the distance between a point on one wave and a corresponding point on the next wave, e.g., the distance between the crest of one wave and the crest of the next wave. The nominal wavelength of red light is taken as 700 nm; for violet light, 400 nm.