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Darkfield microscopy is a specialized illumination technique that benefit from the oblique lighting to improve contrast in samples that are difficult to see clear under normal lighting conditions in light field.
All of us are very familiar with the appearance and visibility of the stars on a dark night, despite its enormous distances from the earth. The stars can be seen by the sharp contrast between their dim light and the black sky.
This principle is applied in dark field (also called dark background) in microscopy. It is a simple and popular method to make unstained objects clearly visible. These objects usually have refractive indexes very close to the value of their environment and it is difficult to see them in conventional light field microscopy. For example, many small aquatic organisms have a refractive index between 1.2 and 1.4, resulting in a negligible optical difference from the surrounding aqueous medium. These are the ideal candidates for darkfield illumination.
Darkfield illumination blocks the passage of the central light, which normally passes through and around the sample, allowing oblique rays to pass through all angles and directly to the sample. The upper lens of a simple darkfield Abbe condenser is spherically concave, allowing the rays of light emerging from all angles of its surface to form an inverted cone of light with a vertex centered on the plane of the sample. If there is no sample and the numerical aperture (N.A.) of the condenser is greater than that of the objective, oblique rays intersect and do not enter the objective, due to their obliqueness. The field of vision is dark.
The objective / condenser (special) illustrated in Figure 3 has a large N.A. which represents the most sophisticated darkfield microscopy. The objective contains an internal iris diaphragm that is used to reduce N.A. of the objective at values below the inverted light cone emitted by the condenser. The design of the darkfield condenser depends on the internal mirrors that project an aberration-free light cone onto the sample plane.
When a sample is placed on the preparation, especially without dyeing, the oblique rays cross the sample and are diffracted, refracted and / or reflected by optical discontinuities (such as cell membrane, nucleus and internal organelles) allowing these faint rays to penetrate the objective. Then the bright organisms appear on a dark background in the sample.
Ideal candidates (samples) for darkfield illumination include many aquatic living organisms, diatoms, small insects, bone, hair, unstained bacteria, yeasts and protozoa. Non-biological samples include minerals and chemical crystals, colloidal particles, counting dust samples, and thin sections of polymers and ceramic materials that contain small inclusions, porosity differences, or refractive index inclinations. Care should be taken in preparing samples for darkfield microscopy due to the characteristics that are above and below the focus plane that can also disperse the light and contribute to image degradation. The thickness of the sample and the thickness of microscope slides are also very important and, in general, a thin sample is ideal to eliminate the possibility of diffraction objects that can interfere with the formation of the image.