Optical Contrast Methods

Brightfield microscopy often only provides a weak image of unstained specimens, in which only a few details are detectable. A stronger contrast is important to see more details in the picture. One way to enhance contrast is to stain the sample. As this is not possible for living organisms, however, they stay uncolored and appear inconspicuous. These specimens actually interact with the incident light in an optical microscope as well. Yet a phase shift occurs that is not detectable with the human eye. The eye can only experience changes in amplitude (brightness) and in frequency (color).

Contrast refers to the possibility to distinguish between the sample and the background and to discover details in the sample. It is defined as the difference in light intensity between the image and the adjacent background compared to the overall background intensities. For the human eye these differences need to be at least two percent to be detectable. Great improvements may be achieved using photo detectors.

However, the contrast is not only determined by the specimen and its interaction with light itself. The optical system which is used to observe the specimen and its ability to record the image information is critical as well. In a microscopic system, contrast depends on the right aperture settings, the grade of the optical aberration, the contrast method used, the specimen and the detector.

The contrast of an image acquired with an optical microscope can therefore be influenced independently of contrast methods by changing the aperture settings of diaphragms. But if for example the condenser is stopped down too far, the resolution will be impaired and diffraction artifacts may appear.

The oldest method of achieving adequate contrast is probably to stain the sample first. Commonly, this is only possible for dead material and occasionally requires some complicated staining protocols. If one wants to observe living cells, staining is not easily done. Therefore different techniques – provided by an adequate optical microscope – aim to change the phase shift, which is caused by the interaction of light with the specimen, into an amplitude shift.

Phase contrast and differential interference contrast (DIC) are examples of these contrast methods. Other optical contrast methods are darkfield and polarization contrast.

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