Phase contrast microscopy

The phase contrast method is a method of imaging in light microscopy. This takes advantage of any change in addition to the amplitude and the phase of light waves when passing through a medium depends on its refractive index. Thus, a direct mapping of structures is possible that only have a low self- contrast and would be visible in brightfield microscopy with artificial coloring.

Widely used phase contrast microscopy is the investigation of biological samples that differ only slightly in their density. The phase contrast method was developed in 1932 by the Dutch physicist Frits Zernike and 1941 introduced by the Jena works in the microscopic field. In 1953, Zernike for his discovery of the Nobel Prize for physics.

Contrast in light microscopy

The perceptibility of details in an image depends on the resolution and the contrast of the image. In particular, the light microscopy of biological objects is affected in many cases by poor contrast of the images produced, namely whenever the objects appear by general light absorption ( darkening ), by spectrally specific light absorption (intrinsic colors) or by very large differences in refractive index with sufficient contrast. Therefore, there are various methods to increase the contrast. For example, individual objects or their components are specifically stained with dyes. Another method is to reduce the illumination, by narrowing the iris diaphragm of the illumination apparatus ( the condenser ). This object parts are represented with different refractive index with higher contrast. But this method has the serious drawback that the resolution of the image is greatly reduced, since the resolution of the microscope depends on the numerical aperture of the object illumination. In addition, the representation of living biological material is possible only with difficulty by artificial coloring, as many cells perish by the dye.

Principle of the phase-contrast method

The phase-contrast method by Frits Zernike takes advantage of differences in the refractive index and the thickness of the object to produce a light-dark contrast, without reducing the illumination aperture and thus the resolving power of the microscope important. Since light propagates in media with different refractive indices at different speeds, resulting as it passes through an object, which is optically denser than its surroundings, a phase difference with respect to the light that does not pass through this object.

In order to view this phase shift in brightness differences, a phase ring in the objective as well as a ring diaphragm in the condenser are installed in a phase contrast microscope. The annular orifice restricts the light incident on the sample at a particular angle. No preparation is under the microscope, meets the entire sunken by the annular aperture background light on the phase ring. This consists of a material which attenuates the light, and its phase shift at the same time by 90 °.

However, a preparation example of a sample with transparent cell brought into the beam path, the light is deflected by the diffraction part to the structures of the cells. However, this object diffracted light passes in contrast to the non-diffracted light mainly not by the phase ring and is accordingly not affected by this. However, the diffraction in the sample also causes a phase shift depending on the refractive index.

In the image plane, it can start to interference between background and object light. This maximum contrast is obtained, the phase shift of the background light in the phase ring must be moved so that the background light with the object light as much as possible this weakens with interference. The phase ring in the objective must therefore be sized approximately corresponding to the most abundant light refractive indices and thicknesses of the objects under consideration. This causes the object now appears mostly dark in the light. This is known as positive phase contrast. For objects with a particularly high refractive index (eg, endospores of bacteria) proposes to contrast and they are brighter than the background.

In a rare procedures applied to the phase ring is sized so that even with usual objects results in a reversed contrast, so that they appear bright on a darker background; This is referred to as a negative phase contrast.

By using the phase contrast method, the resolution of a microscope does not improve over the diffraction limit.

• Comparison of different microscopy methods on a sample tissue paper.

Bright field microscopy, contrast is generated by absorption of light in the object.

Polarization microscopy, contrast is generated by rotation of the polarized light in the sample.

Dark field microscopy, contrast is generated by the scattered light in the object.

Applications

Most often, the phase-contrast method is used in light microscopy of biological objects. In particular, in the observation of cells that are virtually invisible in normal light microscope, to contrast images resulted without the need for staining.

The pollen analysis of even the finest structures of the surface of pollen grains can be visualized with the aid of the phase contrast method.

Furthermore, the image formation is based on the high-resolution transmission electron microscopy and high-resolution bright-field scanning transmission electron microscopy on the effect of phase contrast - but the case of the wave functions of the electron beam.