Optical microscope

(optics) An instrument used to obtain an enlarged image of a small object, utilizing visible light; in general it consists of a light source, a condenser, an objective lens, and an ocular or eyepiece, which can be replaced by a recording device. Also known as light microscope; photon microscope.

An instrument used to obtain an enlarged image of a small object. In general, a compound microscope consists of a light source, a condenser, an objective, and an ocular or eyepiece, which can be replaced by a recording device such as a photoelectric tube or a photographic plate. The optical microscope is limited by the wavelengths of the light used and by the materials available for manufacturing the lenses.

Magnifying power

The quality and design of the lens system determines the magnifying power, details of image formation, and color correcting capabilities of a light microscope. The magnifying power of a compound microscope is the product of the magnification of the objective and the magnifying power of the eyepiece. The latter is computed like that of any magnifier. The magnification of the objective is equal to the distance from the second focal point to the image formed by the objective, divided by the focal length. An objective of 18-mm (0.7-in.) focal length thus has a power of 10×. It is customary to specify objectives in terms of magnifying power instead of focal length. The distance mentioned is called the optical tube length (generally 180 mm or 7 in.), and is to be distinguished from the mechanical tube length, which is the length of the mechanical tube itself. See also Magnification.

Catadioptric systems

Catadioptric systems have been developed for microscopes. Their great advantage is their comparatively small chromatic aberration. Pure mirror systems have no color aberrations. In catadioptric systems, therefore, it is customary to assign all the power to the mirror or mirrors, keeping the refracting system nearly afocal (Fig. 1a). The chromatic errors of the entire system remain small, and the refracting part can be used to correct the remaining monochromatic errors. All microscopic work in the ultraviolet region is done with catadioptric systems (Fig. 1b).

Two types of catadioptric objective. (a) Maksutov type. (b) 53X, NA 0.72, ultraviolet objective, designed by Gray. Glass elements in the latter serve purely as reflectors. (Photographic Service Department, Kodak Research Laboratory)

Condensers

An external auxiliary lens is used to condense the light from a light source so that the object is brightly and uniformly illuminated. The usual purpose of a condenser system is to make sure that as much light as possible coming from the object goes through an optical system. Condensers are used in macroscopic projection, in which an illuminated film or slide is imaged with the help of a projection objective or magnifier. In microscope systems, they are used to direct the light from a light source so that the rays from any object point fill most of the entrance pupil. A condenser system is usually arranged to image the light source onto the entrance pupil of the optical system (Köhler illumination). The condenser is generally corrected for spherical aberration, color, and sine condition, although the requirements are slightly different than in an image-forming system.

Light microscope

The mirror, condenser, oculars, and body tube of the light microscope are frequently known as the optical train. The stand, stage, and adjustments comprise the mechanical part of the microscope.

A mirror is usually attached to the substage of the microscope to reflect light along the optic axis of the microscope. When no condenser is used, the concave mirror is used because it concentrates more light on the specimen; a plane mirror is used with a condenser.

Objectives vary from a simple doublet lens to complex corrected lens systems. Achromatic objectives are corrected for spherical aberration in one color and for chromatic aberrations in two colors. Apochromatic objectives are corrected to focus three colors together and the spherical aberration is minimized for two colors. The resolving power of an objective, the least distance at which two objects can be seen to be separate, is equal to the wavelength of light λ divided by the sum of the numerical apertures of the condenser and objective used. The larger the numerical aperture, the greater is the resolving power. Objectives are described also by the equivalent focal length. Objectives of shorter focal length have less depth of field, less working distance, and greater magnification.

Photomicrographic objectives are designed to produce a flat image with little distortion. For convenience, two to five objectives can be mounted on a revolving nosepiece to be parfocal and parcentric, so that the specimen remains almost in focus at the center of the field as the objectives are changed.

The commonly used Huygenian ocular has a fairly flat field with marked pincushion distortion. Compensating oculars complete the color correction for apochromatic objectives and have less distortion, but they do have curvature of field. See also Eyepiece.

The monocular body tube may be of adjustable length. American microscopes are designed for a mechanical tube length of 160 mm (6.3 in.) and a cover-glass thickness of 0.18 mm (0.007 in.). The draw tube is lengthened for thinner and shortened for thicker cover glasses to correct for the spherical aberrations from cover glasses of incorrect thickness.

Binocular bodies are designed for the use of both eyes. Most binocular bodies use prisms to reflect one-half of the light to each eye. Because each eye sees the same field, these binocular bodies do not give stereoscopic vision. The binocular is often longer than the monocular body and the proper tube length is maintained with a compensating lens.

Inverted microscope

The inverted microscope has the body of the microscope, including the objective and the ocular, below the stage and the illumination above the stage for transmitted light. The inverted microscope is especially useful for the examination of surfaces. Large and awkward specimens can be moved over the stage more readily than with the usual microscope. The inverted microscope is also useful for microdissection and the observation of hanging-drop preparations and is convenient for observing chemical reactions, melting-point determinations, and photomicrography.

Comparison microscope

The comparison microscope is an arrangement of two microscopes connected by a special viewing ocular so that the field of one microscope is seen at one side of a vertical dividing line and the field of the other microscope on the opposite side of the dividing line; or it may be a projection type of microscope in which the image is compared with a template or known pattern.

Dissecting microscope

Dissecting microscopes are of two types. The simplest is a magnifying glass mounted on a support above a glass plate, used for the dissection of materials.

The more usual dissecting microscope, often called a Greenough microscope, is a stereoscopic microscope composed of two separate microscopes fastened together and used as a single unit on one stand (Fig. 2). This is a truly stereoscopic instrument because the right eye sees the specimen from the right side and the left eye from the left side. Prisms are usually included in the body tube to erect the image; thus movements of the specimen are direct and are not reversed as with the monobjective microscope.

Diagram of light rays as they pass through a binocular biobjective microscope.

Metallurgical microscope

The metallurgical microscope is a laboratory microscope with a focusing stage and a vertical illuminator, used primarily for the examination of metal surfaces.

Near-infrared microscopy

Near-infrared microscopy is an optical method that can be used for studying a variety of materials that are opaque in transmitted visible light (400–700 nm) yet translucent in the near infrared (700–1200 nm). The method utilizes the near-infrared optical microscope, a device for the conversion of the near-infrared image to a visible image.

Near-field optical microscopy

A fundamental law of optics, the so-called diffraction limit, states that two objects can be imaged as separate entities only if their distance is larger by about one-half the wavelength of visible light, which ranges from 400 to 700 nm. As a consequence, conventional optical microscopy is restricted to a resolution of about 200 nm, and this is not enough for many important observations. The scanning near-field optical microscope (NSOM or SNOM) circumvents the diffraction limit. In contrast to nonoptical methods of surpassing this limit, such as electron microscopy, it can provide information on such qualities as color, luster, transmissivity, and birefringence, which are sensitive indicators of material composition and status. Furthermore, it operates at ambient conditions, a prerequisite for observations of living organisms. See also Microscope.