What is darkfield microscopy?
The methodology surrounding darkfield microscopy, although widely used for imaging transparent specimens throughout the 19th and 20th Centuries, is limited in use to physically isolated cells and organisms (as presented in Figure 1(e)). In this technique, the condenser directs a cone of light onto the specimen at high azimuths so that first-order wavefronts do not directly enter the objective front lens element. Light passing through the specimen is diffracted, reflected, and/or refracted by optical discontinuities (such as the cell membrane, nucleus, and internal organelles) enabling these faint rays to enter the objective. The specimen can then be visualized as a bright object on an otherwise black background. Unfortunately, light scattered by objects removed from the focal plane also contribute to the image, thus reducing contrast and obscuring specimen detail. This artifact is compounded by the fact that dust and debris in the imaging chamber also contribute significantly to the resulting image. Furthermore, thin adherent cells often suffer from very faint signal, whereas thick plant and animal tissues redirect too much light into the objective path, reducing the effectiveness of the technique.
Recently, a renewed interest in transmitted darkfield microscopy has arisen due to its advantages when used in combination with fluorescence microscopy. Darkfield microscopy is a specialized illumination technique that capitalizes on oblique illumination, as described above, to enhance contrast in specimens that are not imaged well under normal brightfield illumination conditions. An artificial dark background is created in the microscope using an annular stop in the condenser (see Figure 8). Although the condenser optics then illuminate the sample, they do so with a hollow inverted cone of light that passes through the specimen at extremely oblique angles at all azimuths so that only the light interacting with specimen features is able to enter the microscope objective to form a bright image of the specimen superimposed onto a dark background. To create the high angles, it is necessary for the objective aperture to be smaller than the inner aperture of the illuminating light cone (see Figure 8(a)). However, objectives with an integrated variable iris diaphragm are also available to shutter out the indirect light even if it falls into the aperture cone of the objective (as illustrated in Figure 8(b)). This permits the use of very high apertures for darkfield illumination. Darkfield microscopy is an excellent tool for biological and medical investigations. It can be effectively used at high magnifications to image living bacteria, or at low magnifications to view and image cells, tissues, and whole mounts.