Light Microscopy – Brightfield and Darkfield Illumination

Abstract

From the one specimen, more than one type of image can be obtained in the microscope. Worrying though this may seem, this knowledge can be used to great advantage by the discerning microscopist. Resolving power (minimum resolved distance) always remains a major consideration, but for the biologist, poor contrast is often a detriment to satisfactory observation, and contrast improvement has been a major source of development in microscope technique. At its simplest, contrast, is profoundly influenced by the way that light is directed on to the specimen, an advantage that is made use of in darkfield illumination.

In general, the illumination for the microscope should have an adequate intensity, provided through a separate intensity control. The observed field should be completely and evenly illuminated. Controls for the area of illumination and the angle of illumination should be learned to maximize microscope performance.

Key concepts:

  • A research microscope usually has at least two iris diaphragms in the light path. Their separate functions must be recognized.

  • Light intensity should be controlled by a transformer, not the condenser iris diaphragm.

  • For measurement, a scale inserted in the eyepiece can be superimposed on the microscope image.

  • No one set of instructions will explain the workings of all models of microscopes.

  • For many biological applications poor contrast of the image is limiting to satisfactory observation.

  • Brightfield (Köhler) Illumination – Theory.

Keywords: brightfield; darkfield; darkground; filters; Köhler illumination; Rheinberg illumination

Figure 1.

Köhler illumination. The diagram on the left highlights the image‐forming ray path that incorporates the four conjugate planes associated with the specimen plane. On the right, the illuminating or aperture ray path is emphasized – four conjugate planes that incorporate the filament and the lens ‘apertures’.

Figure 2.

Plasmodium vivax in a human blood smear; brightfield (left) and darkfield (right). The bar represents 10 μm.

Figure 3.

Equipment for darkfield microscopy. From the left: a patchstop, a dry darkfield condenser, an immersion darkfield condenser and an oil‐immersion objective with iris diaphragm.

Figure 4.

A blood smear with Trypanosoma sp.: brightfield (left) and darkfield (right). The bar represents 10 μm.

Figure 5.

(a) A normal brightfield, but very poor contrast image of diatoms; (b) a homemade Rheinberg filter and (c) the Rheinberg image of the diatoms.

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References

Bradbury S and Bracegirdle B (1998) Introduction to Light Microscopy. Oxford: BIOS Scientific.

Bradbury S and Evennett PJ (1996) Contrast Techniques in Light Microscopy. Oxford: BIOS Scientific.

Evennett PJ (1993) Depth of field and depth of focus explained. In Queries. Proceeding of the Royal Microscopical Society 31: 64–66.

Oldfield R (1994) Light Microscopy; An Illustrated Guide. London: Wolfe Publications.

Rost F and Oldfield R (2000) Photography with a Microscope. Cambridge: Cambridge University Press.

Further Reading

Köhler A (1893) A new system of illumination for photomicrographic purposes. English translation in (1993). Proceedings of the Royal Microscopical Society 28: 181–188.

Rheinberg J (1896) On an addition to the methods of microscopical research, by a new way of optically producing colour‐contrast between an object and its background, or between definite parts of the object itself. Journal of Royal Microscopical Society Series II XVI: 373–388.

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How to Cite close
Oldfield, Ronald J(Jan 2010) Light Microscopy – Brightfield and Darkfield Illumination. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002995.pub2]