darkfield

Darkfield is an illumination method used in optical microscopy and related imaging techniques to visualize small or transparent specimens. In a darkfield arrangement, direct light is prevented from entering the objective; only light scattered by the specimen is collected. This produces bright objects on a dark background, enhancing contrast for features that are nearly invisible under conventional bright-field illumination.

Principle and instrumentation: A special condenser with an opaque disk blocks the central rays, or an annular diaphragm creates a hollow cone of illumination. When Koehler illumination is used, the effect is uniform. The scattered light from tiny structures, motile organisms, or edges of cells is captured, yielding high-contrast images even when stains are not used.

Applications: Widely used to observe bacteria such as spirochetes, algae, pollen, and living cells; particularly suited to unstained or living specimens; used in microbiology and cytology; can reveal motility and subtle surface features.

Limitations: Not suitable for thick or densely packed samples; lower light efficiency; image brightness can be uneven; requires careful alignment and clean optics; resolution is similar to bright-field but contrast arises from scattering rather than absorption.

Other uses: In electron microscopy, dark-field imaging uses an off-axis objective aperture to collect electrons diffracted by the specimen, producing images that highlight crystalline structures and defects.