As we have already seen at part III of this mini-course, in light microscopy, also called bright field microscopy, the object analyzed shows darkened and the surrounding environment shows very bright. In dark field microscopy, the opposite occurs, i.e. the object shows very bright, in contrast with the dark background.

In order to fulfill its purpose, the technique of dark field microscopy makes use of a special type of condenser lens, which makes that only the light that reach an object at the slide enters the objective lens.

If the slide contains just a transparent material, like saline solution or purified water, the light will deviate and will not enter the objective lens. In this case, when looking through the ocular lens, the field will be completely dark.

However, if the slide contains any object with a different refraction index, there will be dispersion of light by reflection and refraction, and the light that reaches the object will enter the objective. In this case, when looking through the ocular lens, the object will appear illuminated against a dark field.

The condenser lens used by the dark field microscopy is called cardioid condenser and there are two types: dry and wet. The wet one requires a liquid medium between the objective and the slide, while the dry one does not. The wet one provides clearer images, in opposite of the dry one. For this reason, the most frequently used type of condenser is the wet one and the liquid used between the slide and the objective is the immersion oil.

The dark field microscopy is particularly useful for the analysis of not stained cells, components or microorganisms. An example of application is the observation of bacteria that cannot be appropriately stained by conventional means, like Borrelia burgdorferi (figure above), which infects a special kind of tick and causes the Lyme disease in humans.

Next article: ultra-violet microscopy.