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Optical Microscopy 1. Resolution 2. Visability 3. Special Subjects 4. Glossary 5. References |
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| Optical Microscopy 3. SPECIAL SUBJECTS Condenser The optical system, usually directly below the object, which illuminates the object. Years ago, electrical/electronic engineers renamed their condensers 'capacitors'. That did not happen in optics. Cover glass, cover slip The object being examined is often covered with a thin sheet of glass. Because the glass is part of the optical system, its thickness is important for lenses used dry, with high numerical aperture and high magnification. The nominal thickness is 0.17 mm, and cover glasses are classified by thickness as #1, #1 ½, and #2. Some high-power dry objectives have correction collars for adjustment for the cover glass thickness, and very careful microscopists may measure the cover glass thickness. Some objectives are made for use without cover glasses. Dry / Immersion Lenses corrected for 'homogeneous immersion' are made to be used with an immersion medium (instead of air) between the condenser and slide, slide and coverslip, and coverslip and objective. The immersion liquid is usually an oil with refractive index 1.515, essentially the same as optical glass. Lenses are also made for immersion in water, glycerin, methylene iodide, or other liquids of high refractive index, but those are not 'homogeneous' immersion systems. A 'dry' lens is not designed for immersion; resolution is not quite as good but often entirely adequate, and a dry system is much easier to use. Illumination: Koehler, Nelson, Abbe, critical It is important that the object be illuminated brightly, evenly, and at high numerical aperture. With Nelson (Abbe, critical) illumination, the condenser is adjusted to focus the light source (such as an oil flame) onto the object. Most electrical lamps for microscopes have coiled filaments, and focusing those onto the object gives very uneven lighting, although there are 'ribbon' filament lamps designed to avoid that problem. Koehler illumination uses a lens immediately in front of the lamp, and focuses an image of that lens onto the object. Some systems place a diffuser (ground glass) in front of the lamp instead of a lens; this is generally less desirable, but cheaper. Infinity correction Objective lenses are commonly designed to focus an image at the distance of the eyepiece focal plane ('optical tube length'). If accessories are placed between objective and eyepiece, they must keep the focus at the same place. That often requires extra lenses. 'Infinity-corrected' objectives focus the image only at infinite distance (i. e., light rays are parallel). At or near the objective, another lens focuses the parallel rays at the eyepiece focal plane. Optical tube length is not critical in these systems. Magnification Visual magnification = objective magnification x eyepiece (ocular) magnification (and sometimes x body tube magnification). For prints or projection also multiply by the photographic or projection magnification. Monocular / Binocular / Trinocular / Stereo Monocular: has only one eyepiece (ocular lens); used with one eye. Binocular: two separate eyepieces, one for each eye. Trinocular: binocular eyepieces plus a separate tube for a camera. (There are also systems with one eyepiece and a separate tube for a camera, and 'observation tubes' made so that a student and teacher can see the same image at the same time.) A stereomicroscope has two separate light paths, one for each eye, including two objective lenses (perhaps two separately ground parts of a single lens). A stereomicroscope must be binocular or trinocular, but a binocular microscope is often not stereoscopic. Mounting medium The substance in which the object is immersed (in a thin layer) on the microscope slide, normally covered by the cover slip. It is sometimes water, but media of high refractive index are particularly useful because a large difference in refractive index improves visibility. Examples: immersion oil (n = 1.515); Aroclors (polychlorinated biphenyls, n about 1.66); Hyrax; Styrax; Naphrax (tradenames); arsenic disulfide (n » 2.3). Nicol / Polaroid Polarizing microscopes were first made with Nicol prisms (of calcite) to select polarized light. Essentially all now use 'Polaroid' type films which typically contain oriented crystals of quinine sulfate (herapathite), or something of that sort. Parfocal It is convenient to mount several objective lenses of different magnifications (e. g., 10x, 40x, 100x) on a rotating nosepiece. If they are 'parfocal', very little change of focus is required when changing lenses. Particle sizes ('Horizontal' and 'vertical' refer to the apparent position of the particle in the field of view.) Feret's diameter: distance between two vertical lines tangent to the ends of the particle. Martin's diameter: length of the horizontal line that appears to divide the particle into halves. Maximum horizontal intercept: longest horizontal line in the particle. Projected area diameter: diameter of a circle which appears to have the same area as the particle (estimated using a reticle showing a series of circles of known sizes.) Photomicrography / Microphotography / Photomacrography Photomicrography : making enlarged images of small objects using a microscope. Photomacrography : making enlarged images with an objective lens only (to about 50x). Microphotography: making very small images (microdots, microlithography for solid-state circuits). Refractive index, Becke line, dispersion staining When a transparent microscopic object is in a liquid with a different refractive index (RI), a slight halo can be seen at the edge of the object. The halo (Becke line) moves toward the medium of higher RI as the object is moved away from the objective lens. The RI of the object can be estimated fairly accurately by using a series of liquids of known RI and finding the one in which the Becke line does not appear. Because RI of the liquid varies with wavelength (dispersion), the Becke line may have colored edges. Dispersion staining is a system for emphasizing those colored edges by reducing the numerical aperture ('annular stop') or by blocking the center of the illuminating beam ('central stop'). It is particularly useful in particle identification. Another method of checking particle RI uses oblique illumination. Simple / compound microscope A simple microscope is a magnifier with a single lens (perhaps with several components). A compound microscope -- usually just a 'microscope' -- has a second lens system (ocular, eyepiece) which magnifies the intermediate image produced by the first lens system (objective). Stage micrometer / ocular (eyepiece) micrometer The eyepiece micrometer superimposes a scale or a movable line (filar micrometer, screw micrometer) in the field of view for measurements of the object. It is calibrated using a stage micrometer, which is a microscope slide with an engraved scale, typically 1 mm long divided at 0.01 mm (10 µm) intervals. The stage micrometer may be traceable to a standard maintained by the (US) National Institute of Standards and Technology (NIST). Abbreviations / designations Most of these need not be translated. Most of the chemicals named are fluorescent stains.
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