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dark field illumination

What is dark field illumination?

Darkfield Illumination is a transmitted light technique that uses oblique light to illuminate the sample. Light that does not impinge on the sample is not collected by the objective and results in a dark background. Light that interacts with the sample is scattered (refracted, reflected, and/or diffracted) and is “bent” toward the objective collection angle. This light is collected by the objective and is seen as light spots or areas (resulting from scattered light) on a dark background. At a first sight, images captured using dark field illumination seem unusual to the viewer. The light shines at a shallow angle. According to the principle “angle of incidence = angle of reflection”, all the light is directed away from the viewer (the camera), the field of view therefore remains dark.Inclined edges, scratches, imprints, slots, and elevations interfere with the beam of light. At these anomalies, the light is reflected towards the camera, or mostly only strayed. These defects appear bright in the camera image in this way.

Contrast is therefore generated and the sample visualized.Darkfield illumination is provided to the sample by a specialized condenser. The simplest DF condenser has a Stop, or Annulus illuminating ring (A). Here, an opaque circle obscures the central portion of the condenser light path. This allows only light in a ring to illuminate the sample.

The diameter of the central stop, and thus illuminating annulus, is such that the angle of light is greater than the collecting angle of the objective. Thus without a sample, no light is collected by the objective. This kind of DF stop is useful only for low magnification objectives (<20x).For higher magnification objectives, modifications of the Annular Stop are: B: Immersion paraboloid; C: immersion double mirror concentric; D: cardioid concentric. Gray cone represents the light reflected and refracted from the specimen and collected by the objective. Hatched areas represent glass. Light blocking stops (s) limit light transmission to a hollow cone. i: Immersion oil.; r: reflecting surfaces. (Ruzin,1999).The diameter of the central stop is dependent on the NA of the objective.

Slightly oblique illumination

This scheme illustrated here already shows the gradual transition from bright field to dark field illumination. Due to stray light effects caused by the texture, the surface is is still sufficiently bright that the object is easy to recognise. However, inclined edges and notches are brighter than the remaining image areas, which is typical for dark-field illumination.

Light inciding at very shallow angles

The scheme illustrated here shows the effect of a very strong dark field. The light shines on the surface at an extremely shallow angle. Small scratches, scuffs, dust particles, and even fingerprints on polished surfaces can be made visible, while the surface appears very dark.Extreme dark-field illumination should be positioned very close to the surface. Working distances from 0.5 to 2 cm are maximally possible. If the distances are greater, the surface is no longer illuminated.

 

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