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Phase Contrast

The method of Phase-Contrast microscopy was invented by Frits Zernike (who received the Nobel Prize for this invention) in 1953. The method is to increase the contrast and make subjects so visible transparent and colorless, which in this way may differ from their surrounding medium (eg water, or the means of mounting), because they will be to have a different refractive index. Using bright field microscopy, these objects are almost invisible. The optical phase contrast microscope is able to convert the differences in refractive index to a difference in brightness. The optical effect that is used is in a phase shift into the beam. Waves during their passage through the cell nuclei, cytoplasm or water, are slightly out of phase, because the media have slightly different refractive indices. Since the higher the refractive index of a medium, the lower the speed of light in the medium itself, a light wave passing through a nucleus of a cell is far behind regard the light waves passing through the surrounding water. The amount of this "delay" is called the phase difference. Before crossing the sample, the waves are in phase with each other, but not any more after going through various materials. The length of the route and type of the medium determine the relative magnitude of the phase difference in light passing through the preparation.
As we know the human eye can not see these microscopic phase shifts in the image, this provides the optical system of phase-contrast microscope, which transforms the "late" phase in levels of gray.
The working of a phase contrast microscope is similar to that in dark field, in this case, the "stop" in the filter holder of the condenser is replaced by an annular diaphragm. An annular diaphragm of the same magnitude is in the objective that has two purposes, reduces the direct light coming from the condenser and introduces an additional phase shift in it. The objectives used in the phase contrast microscope, therefore, are special objectives, with a phase ring around the pupil. They are recognizable because they are marked with the symbol "Ph1", "Ph2" and "Ph3" depending on the magnification factor, usually "Ph1" for magnifications up to 10x, "Ph2 for magnifications up to 40x," Ph3 "by 40x onwards. Such objectives can also be used for observations in bright field, with a slight drop in image quality.
The condenser is equipped with phase rings Ph1, Ph2 and Ph3 to be used depending on the objective chosen.
An operation to be performed with this technique is the "centering" of the annular aperture in the condenser, so that the image in the optical path of the annular diaphragm coincides exactly with the position of the ring phase. To help to obtain a very precise centering, using the "eye-phase" or a "Bertrand lens, which are inserted into the tube instead of the eyepiece itself and are to align the phase rings, the centering of the condenser is carried out with two adjustments on the sides of the same.
A phenomenon to be highlighted because it is annoying in the phase-contrast is the halos of light that form the edges of the structures. They are caused by an optical effect and may lead to the "unreadable" images, especially with thick samples, since the halos overlap several times. For this reason, the phase contrast is recommended only with very thin and possibly little aggregate objects.
Depending on the system used, samples will be highlighted as bright on a dark background (negative phase contrast) or dark on a bright background (positive phase-contrast).
The phase-contrast microscopy is commonly used for observation of bacteria, protozoa, micro-organisms and all the subjects you want to observe without the aid of a staining.