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dark field microscopy

What Principles of dark field microscopy?

What Principles of dark field microscopy?

To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye. Instead of coming up through the specimen, the light is reflected by particles on the slide. Everything is visible regardless of color, usually bright white against a dark background.

Pigmented objects are often seen in “false colors,” that is, the reflected light is of a color different than the color of the object. Better resolution can be obtained using dark as opposed to bright field viewing.

Sophisticated equipment is not necessary to get a dark field effect, but you do need a higher intensity light, since you are seeing only reflected light. At low magnification (up to 100x) any decent optical instrument can be set up so that light is reflected toward the viewer rather than passing through the object directly toward the viewer.

dark field microscopy

When to Use a dark field microscopy?

Dark field microscopes are used in a number of different ways to view a variety of specimens that are hard to see in a light field unit. Live bacteria, for example, are best viewed with this type of microscope, as these organisms are very transparent when unstained.

There are multitudes of other ways to use dark field illumination, often when the specimen is clear or translucent. Some examples:

Dark field illumination of caffeine crystalsLiving or lightly stained transparent specimens
Single-celled organisms
Live blood samples
Aquatic environment samples (from seawater to pond water)
Living bacteria
Hay or soil samples
Pollen samples
Certain molecules such as caffeine crystals (right)
Dark field microscopy makes many invisible specimens appear visible. Most of the time the specimens invisible to bright field illumination are living, so you can see how important it is to bring them into view!

dark field microscopy

dark field microscopy at High Magnifications

dark field microscopy at High Magnifications

For more precise work and blacker backgrounds, you may choose a condenser designed especially for darkfield, i.e. to transmit only oblique rays. There are several varieties: “dry” darkfield condensers with air between the top of the condenser and the underside of the slide–and immersion darkfield condensers which require the use of a drop of immersion oil (some are designed to use water instead) establishing contact between the top of the condenser and the underside of the specimen slide. The immersion darkfield condenser has internal mirrored surfaces and passes rays of great obliquity and free of chromatic aberration, producing the best results and blackest background.

Perhaps the most widely used darkfield condenser is the paraboloid, consisting of a solid piece of glass ground very accurately into the shape of a paraboloid, as illustrated in Figure 5(b). Light incident upon the reflecting surface (between the glass and condenser housing in Figure 5(b)) of a paraboloid condenser will be focused at the focal point of the reflector. Most paraboloid condensers are cut to ensure that the focal point is slightly beyond the top of the condenser so that parallel light rays will be focused at a position that maximizes illumination of the specimen. The light stop at the bottom of the glass condenser serves to block central rays from reaching the specimen. Light rays that are reflected by the condenser are angled higher than the critical angle of reflection and converge at the principal focus of the condenser. The combination of a glass slide, mounting medium, and immersion oil (between the condenser and the microscope slide) complete the optical homogeneity of the paraboloid shape.

As discussed above, the dry darkfield condenser is useful for objectives with numerical apertures below 0.75 (Figure 5(a)), while the paraboloid and cardioid immersion condensers (Figures 1 and 5(b)) can be used with objectives of very high numerical aperture (up to 1.4). Objectives with a numerical aperture above 1.2 will require some reduction of their working aperture since their maximum numerical aperture may exceed the numerical aperture of the condenser, thus allowing direct light to enter the objective. For this reason, many high numerical aperture objectives designed for use with darkfield as well as brightfield illumination are made with a built-in adjustable iris diaphragm that acts as an aperture stop. This reduction in numerical aperture also limits the resolving power of the objective as well as the intensity of light in the image. Specialized objectives designed exclusively for darkfield work are produced with a maximum numerical aperture close to the lower limit of the numerical aperture of the darkfield condenser. They do not have internal iris diaphragms, however the lens mount diameters are adjusted so at least one internal lens has the optimum diameter to perform as an aperture stop.

Table 2 lists several properties of the most common reflecting high numerical aperture darkfield condensers. This table should be used as a guide when selecting condenser/objective combinations for use with high numerical aperture darkfield applications.

dark field microscopy

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