Contrast methods in microscopy
In standard microscopic observation, a transparent specimen is usually observed in transmitted light. This is also called brightfield microscopy if you want to distinguish it from the following contrast methods.
For dark field, the beam path of the microscope is directed by a special condenser in such a way that the light shines through the specimen obliquely, but without interaction with a specimen no direct light falls into the objective. Only where the specimen (cell structures) diffracts the light, it does enter the objective. The sample therefore appears bright against a black background. Dark-field condensers have either a central aperture that blocks direct light or an elaborate optical construction of lenses and mirrored elements, which is called a cardioid condenser.Je nachdem, welches Objektiv genutzt werden soll, wird ein "trockener" oder ein "Öl"
Depending on which lens is to be used, a "dry" or an "oil" dark field condenser ("oil" for all objectives with oil immersion, usually the 100x objective) is required. If all microscope objectives are to be used for dark field observations, both condensers must be purchased. "Dry" dark field condensers are more versatile (and easier to adjust even for inexperienced users) because they allow a wider magnification range to be used. In combination with a 60x lens, magnifications of up to 900x can be achieved. In the case of oil immersion, the condenser must always also be immerged; this applies in bright field as well as in dark field.
Typical applications for dark field microscopy
Dark field microscopy is particularly suitable as a contrast method when transparent cells or microorganisms that have little contrast will be observed. Typical examples are amoeba or epithelial cells, e.g. of the oral mucosa. Fixing and subsequent colouring is not necessary. Dark field microscopy is also particularly suitable for live cultures of aquatic organisms or algae, which one would like to see in motion, which usually prohibits staining. In addition to the contrast gain achieved, dark field observations are also aesthetically pleasing!
In oblique illumination, a gain in contrast is achieved by covering part or half of the normal illumination beam path. Commercial solutions for this contrast method are unfortunately rare, but this effect can be achieved with a self-made shutter / slider added in the filter level of the condenser. Often, an existing filter holder or an auxiliary lens on the condenser can also be partially swivelled into the beam path. The frame of the filter holder or field lens then blocks part of the light. The preparation is then radiated through at an angle, resulting in a shadow cast on the fine structures of the preparation.
The phase contrast method uses a combination of a special condenser with exchangeable apertures (these generate a light ring) and corresponding phase contrast lenses (with phase ring). The light that radiates through the preparation is diffracted there and undergoes a phase shift on components with different refractive indices. Light that is not diffracted is blocked at the phase ring.
Usually separate phase contrast sets are offered. It is not recommended to equip the microscope only with phase contrast objectives, because although the resolution of the objectives is not impaired by the phase ring, the contrast is worse than with normal bright field objectives. These lenses are also not suitable for polarization.
A phase contrast condenser must be carefully centered so that the light ring and phase ring are projected onto each other. Therefore a change between phase contrast condenser and light or dark field condenser is time-consuming. However, individual bright-field lenses can be used in the revolver in addition to phase lenses, because the phase contrast condenser has a port for bright-field. The phase contrast set contains an auxiliary eyepiece (also known as an "adjusting telescope") for centering or height adjustment of the condenser, so that it can be focused on the planes of the phase and light ring.
The requirements for phase contrast are ideally fulfilled only for green light. Therefore an appropriate green filter is helpful. Since mainly colorless objects are observed in phase contrast anyway, coloring of the resulting image is acceptable.
Typical applications for phase contrast
Similar to dark field, phase contrast is suitable for objects that are colorless and have little contrast. These include, for example, plankton organisms or activated sludge. Cell cultures or cells in the urine sediment can also be better visualized with phase contrast and thus be evaluated more quickly and reliably.
By polarization, optically active or birefringent specimen structures can be highlighted. These can be minerals, but also many plastics or natural materials such as starch etc. show this effect. It is less known that interesting structures can also be highlighted in living organisms, e.g. muscle fibers of daphnia or rotifers. In industry, polarization is mainly used to characterize materials. If the layer thickness of the sample is known, the resulting interference colours can also be used to determine the type of material. In materials research, polarization is also used, for example, to investigate tension in the material by stress birefringence. Especially injection moulded parts or plastic foils or fibres can be examined for manufacturing defects. The classical application is of course geology / mineralogy, in which thin sections of rock are examined in polarized light.
Due to the spectral distribution, halogen illumination is more suitable than LED in polarisation microscopy.