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3. Special Subjects
No matter how good the lenses, clear colorless objects are not very visible. Increasing the visibility of objects under the microscope has followed various routes. Several of these techniques make use of the fact that light is deflected, reflected, or diffracted at boundaries between two materials with different refractive indices.
Primarily for biological subjects. Colored materials (stains) are applied to (or produced in) cells and tissues, or in thin tissue slices cut with a 'microtome'. Different cell structures absorb the light differently. Many stains have been developed, but most do not stain living cells. Those which do are 'vital' stains. Observation is normally with bright-field transmitted light for biological subjects, although metal surfaces can be differentially etched or stained for viewing by incident light (metallography).
Here the object is illuminated by light at a large angle from the optical axis, so that the light does not enter the objective unless it is reflected or refracted by the object (often because of a refractive index difference). Structure in the object appears bright on a dark background. Either incident or transmitted light may be used. Particles smaller than the resolution of the microscope can be detected (but not imaged) with very intense dark-field illumination ('ultramicroscopy').
Somewhat related to dark-field: light near the optical axis, and light at a large angle to the optical axis, pass through differently colored filters to produce an image in two colors.
The object may itself be fluorescent, or it my have been stained with a fluorescent dye (fluor, fluorphor, fluorophor). Enzyme activity can be detected by using a 'fluorogenic' substrate which is not itself fluorescent, but which the enzyme converts to a fluorescent material. The object is illuminated with ultraviolet or blue light, and emits light of longer wavelength (blue, green, yellow, red, or even infrared). Primary filters (excitation filters) are required to keep visible light from the source out of the rest of the system. The source must be relatively intense because fluorescence is usually weak. The most common source is a mercury arc lamp, but tungsten-halogen lamps can be used for fluors which are excited by blue light. Secondary filters (barrier filters) are needed to keep the intense ultraviolet illumination from the eye or camera. A heat filter at the source is usually required. Transmitted or incident, bright or dark field illumination, with incident probably best. Most fluors fade as they are destroyed by the ultraviolet radiation.
Phase contrast (Zernike)
When light passes into a medium of different refractive index, some light is slightly changed in direction (diffracted) with a small change in phase. The undiffracted light passes through a lens coating which increases the phase difference, while the diffracted light misses that part of the lens. After the light rays are recombined in the image, the phase difference causes interference. In a 'positive' phase contrast system, objects of higher refractive index appear darker. The technique is mostly limited to biological work with living cells or tissues, which usually cannot be stained. Green light is normally used. (Note that these descriptions are very brief; Zernike was awarded the Nobel Prize in Physics in 1953.)
Modulation contrast (Hoffman)
As in phase contrast, some light is diffracted at the object. Diffracted and undiffracted light pass through separate filters which cause strong, moderate, or no absorption of light. The final image shows refractive index changes in shades of grey.
Light passing through an object having a higher refractive index than the medium surrounding it moves more slowly. When that light is combined with light which did not pass through the object, there is a phase difference that causes interference, producing dark and bright bands, or color differences, in the image. Incident-light interference can be used to examine surface topography. A 'differential interference contrast' system (DIC, Nomarski) compares light rays passing through adjacent parts of the object. It is sensitive to small changes in refractive index.
This is one of the best means for identifying particles and fibers (including asbestos). Light from the illuminator is 'polarized', originally by a Nicol prism, now usually by a polarizing film, so that all the light vibrates in one plane. If the object is crystalline with its principal planes at angles to the plane of vibration, the polarized light appears to be split into two portions (vectors) which move along the crystal planes. Most crystals have units spaced differently along those planes ('anisotropic'). That causes different refractive indices for light traveling along those planes ('birefringence'), and the portion of the light vibrating in the higher-index plane travels somewhat more slowly than the other portion. When the two parts of the light are recombined in the 'analyzer' (another polarizing film), some wavelengths interfere with each other and are subtracted. Removal of those wavelengths from white light leaves colored light, and the object is seen in color. The refractive index of the object can be estimated from its color by comparison with a 'Michel-Levy' chart. Various accessories ('compensators') can be used for other observations. Some crystallographic measurements are made by inserting a 'Bertrand lens' into the optical path to view the rear focal plane of the objective ('conoscopic observation'). Because mechanical strains in optical glass cause birefringence and would introduce extraneous color, lenses for polarized light microscopy should be 'strain-free'. Transmitted illumination is most common, but some observations can be made with incident light. Polarized light microscopy does not require optical activity or optical rotation in the object.
Reduced numerical aperture
Reducing the numerical aperture of the system, usually by closing down the condenser aperture diaphragm, increases contrast in the image, but reduces resolution. If it is overdone, as it often it, it may introduce apparent structure that does not really exist.
Optical microscopy generally requires very thin specimens (ca. 10 Ám) so that observation of one layer in the object is not affected by absorption, scattering or fluorescence in other layers. With confocal microscopy, light is concentrated onto a point in the object. Light from that point (which may be fluorescent) is focused on a point detector (such as a pinhole in front of a photomultiplier). Layers of the object away from the point being viewed are not illuminated as brightly because the illuminating light is not in focus there; and light coming from those layers is not focused on the detector pinhole. As a result, thin layers of relatively thick objects can be examined. A laser is usually used to provide intense illumination, and, because only one point at a time is illuminated, a scanning system must be used.
Near-field scanning optical microscopy. Actually a means of increasing resolution, it is mentioned here for convenience. With this technique, a laser illuminates a pinhole about 25 - 50 nm in diameter. Scanning the object past the pinhole gives an image in which the resolution is limited by the pinhole size and not by the numerical aperture.
Scanning probe microscopy: various systems using a very small probe which senses surface features, with a video display system . These include atomic force microscopy, in which the probe touches the surface and is moved up or down to follow the topography; thermal force microscopy, measuring surface temperature; lateral force microscopy, measuring friction; magnetic force microscopy, measuring magnetic fields; and electrostatic force microscopy, measuring charge density. Electron microscopy: Transmission electron microscopy (TEM) uses an electron gun instead of a lamp, and electromagnetic lenses, to produce images on film, fluorescent screen, or video system. Scanning electron microscopy moves a fine beam of electrons back and forth across the, measuring back-scattered electrons, current in the object, etc. The electron microprobe is essentially a scanning electron microscope designed to produce and measure characteristic X-rays from the sample.
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