Contrast Enhancement Phase ContrastDIC Nomarski Dark Field

The principle of all contrast enhancement systems is to turn otherwise invisible variations in material (density, polarizing ability, and so forth) into differ-

ences in perceived light intensity. For embryological specimens, some kind of contrast enhancement is usually extremely useful. For example, with fluorescent preparations, standard counterstains may either quench fluorescence or be autofluorescent themselves. There are occasions, however, when a particular contrast enhancement may reduce the information within an image; for example, Nomarski optics may make small labeled objects (such as cell nuclei) less easy to see. It is always worth viewing a specimen both with and without contrast enhancement before photographing. The use of three common image enhancement systems is described below.

3.5.1. Phase Contrast

Phase contrast converts differences in refractive index or thickness within an otherwise transparent specimen into differences in light intensity. This is achieved by illuminating the specimen with a ring of light by means of a phase "annulus" within the substage condenser. The ring of illumination is aligned with a complementary phase plate within the back of the objective. Since these elements must be in register, only objectives of the same geometrical characteristics can be used with a given phase annulus. The phase condenser may therefore contain a number of different annuli for different objectives. The phase annulus (in the condenser) and phase plate (in the objective) are aligned by means of either a focusing telescope (which replaces the eyepiece) or an Amici-Bertrand lens that can be swung into the light path. Phase-contrast optics should be used with thin specimens.

3.5.2. DIC or Nomarski

Differential interference contrast can be used with both thin and thicker specimens to generate a "pseudorelief" image of the preparation. DIC filters enhance local gradients in optical density by prismatically splitting then recombining polarized light. From the lamp, light passes first through a polarizing filter, and then a "Wollaston"prism within or beneath the condenser. Having passed through the specimen, light is refracted by a second Wollaston prism (usually mounted just behind the objective) and filtered by a second polarizing filter, the "analyzer" filter situated closest to the eyepieces. The analyzer is easily swung in or out, and should always be removed for fluorescence imaging, since it reduces the amount of light passing to and from the slide. The polarizer can be rotated to optimize the DIC effect. For good DIC, the field and aperture diaphragms should be optimized for Köhler illumination (see Subheadings 3.4.1. and 3.4.2., Fig. 3A,B).

Since this system utilizes polarized light to generate changes in light amplitude, naturally polarizing media, such as mica and Plexiglas, may generate optical artifacts when placed in the light path. In addition, some biological materials, for example, structures consisting of parallel oriented fibers, are also inherently polarizing or "birefringent." With only the polarizer and analyzer filters of the DIC system present, birefringent structures will therefore appear as either very bright or very dark if oriented at 90° to their inherent optical axis. Birefringence may be useful in identifying particular kinds of structures within a specimen.

3.5.3. Dark Field

Dark-field or oblique illumination is used to observe dark, particulate staining or for example, darkly labeled axons against a homogenous bright-field background. By producing what is effectively a negative image of the specimen, such features become clearer as bright objects against a dark background. The condenser directs an oblique illumination through the specimen, and the objective lens only receives light if it is reflected or refracted by opaque objects in the illuminated field. Where parts of the specimen are transparent, light passes uninterrupted through object space and does not enter the objective. Darkly stained particles or fibers within the specimen interrupt the light path and appear as bright objects.

Certain stains are considerably clearer under dark-field illumination, despite there being no real gain in spatial resolution. However, specimens have to be carefully prepared: the image will be impaired if tissue is not well cleared (the background will not appear black), or if the slide is contaminated with grease or dust (which will be visible as very bright points of light).

Was this article helpful?

0 0

Post a comment