Microradiograph of a 200-/xm-thick section of bone. Black areas are sites of soft tissue, white areas contain high concentrations of calcium salts, and light gray to dark gray areas reflect decreasing amounts of calcium salts. X157. (Microradiograph courtesy of Dr. Jenifer Jowsey.)

companion slide to provide semiquantitative information on the amount of bone mineral in different parts of the ground section.

V MICROSCOPY Light Microscopy

A microscope, whether simple (one lens) or compound (multiple lenses), is an instrument that magnifies an image and allows visualization of greater detail than is possible with the unaided eye. The simplest microscope is a magnifying glass or a pair of reading glasses.

The resolving power of the human eye, i.e., the distance by which two objects must be separated to be seen as two objects (0.2 mm), is determined by the spacing of the photoreceptor cells in the retina. The role of a microscope is to magnify an image to a level at which the retina can resolve the information that would otherwise be below its limit of resolution. Table 1.4 compares the resolution of the eye with that of various instruments.

Resolving power is the ability of a microscope lens or optical system to produce separate images of closely positioned objects

Resolution depends not only on the optical system but also on the wavelength of the light source and other factors, such as specimen thickness, quality of fixation, and staining intensity. With light whose wavelength is 540 nm (see Table 1.1), a green-filtered light to which the eye is extremely sensitive, and with appropriate objective and condenser lenses, the greatest attainable resolving power of a bright-field microscope would be about 0.2 /xm (see page 16 for method of calculation). This is the theoretical resolution and, as mentioned, depends on all conditions being optimal. The ocular or eyepiece lens magnifies the image produced by the objective lens, but it cannot increase resolution.

Various light microscopes are available for general and specialized use in modern biologic research. Their differences are based largely on such factors as the wavelength of specimen illumination, physical alteration of the light coming to or leaving the specimen, and specific analytic processes that can be applied to the final image. These instruments and their applications are described briefly in this section.

table 1.4. Eye Versus Instrument Resolution

Distance Between Resolvable Points

Human eye Bright-field microscope SEM TEM Theoretical Tissue section

The microscope used by most students and researchers is the bright-field microscope

The bright-field microscope is the direct descendant of the microscopes that became widely available in the 1800s and opened the first major era of histologic research. The bright-field microscope (Fig. 1.7) essentially consists of

• Light source for illumination of the specimen, e.g., a substage lamp

• Condenser lens to focus the beam of light at the level of the specimen

• Stage on which the slide or other specimen is placed

• Objective lens to gather the light that has passed through the specimen

• Ocular lens (or a pair of ocular lenses in the more commonly used binocular microscopes) through which the image formed by the objective lens may be examined directly

A specimen to be examined with the bright-field microscope must be sufficiently thin for light to pass through it. Although some light is absorbed while passing through the specimen, the optical system of the bright-field microscope does not produce a useful level of contrast in the unstained specimen. For this reason, the various staining methods discussed earlier are used. Other optical systems, described below, may be used to enhance the contrast without staining.

The phase contrast microscope enables examination of unstained cells and tissues and is especially useful for living cells

The phase contrast microscope takes advantage of small differences in the refractive index in different parts of a cell or tissue sample. Light passing through areas of relatively high refractive index (denser areas) is deflected and becomes out of phase with the rest of the beam of light that has passed through the specimen. The phase contrast microscope adds other induced-out-of-phase wavelengths through a series of optical rings in the condenser and objective lenses, essentially abolishing the amplitude of the initially deflected portion of the beam and producing contrast in the image. Dark portions of the image correspond to dense portions of the specimen; light portions of the image correspond to less dense portions of the specimen. The phase contrast microscope is therefore used to examine living cells and tissues, such as cells in tissue culture, and is used extensively to examine unstained semithin (approximately 0.5-/xm) sections of plastic-embedded tissue.

Two modifications of the phase contrast microscope are the interference microscope, which also allows quantification of tissue mass, and the differential interference microscope (using Nomarski optics), which is especially useful for assessing surface properties of cells and other biologic objects.

scanning beam electron detector

specimen amplifier television screen

projection lens lens specimen

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