Phase contrast microscopy

    Phase contrast microscopy is used for the study of transparent and colorless objects that are invisible in bright-field observation. These are, for example, unstained live animal tissues, some mineral and organic components.

    The essence of the method lies in the fact that a light wave passing through the specimen with incorporated elements having slight differences in refractive indices undergoes different changes in phase (acquires the so-called phase relief). However, phase changes cannot be perceived directly either with the eye or with a photographic plate. To create a contrast image it is necessary to transform the phase changes of the light wave into the amplitude changes (an amplitude relief). To this end, special optics – a phase contrast condenser and a phase objective – are used.

    A phase-contrast condenser is a conventional objective with a revolver and a set of annular diaphragms for each objective. The phase objective is equipped with a phase plate which is obtained by coating the salts of rare earths on the objective. The image of the annular aperture coincides with the ring of the phase plate of the corresponding objective.

   The rays from the illuminator undeflected in the sample and making the image of the diaphragm should entirely pass through the phase ring, which significantly weakens them (it is made absorbing) and change their phase to λ/4 (λ – is a wavelength). And the rays, even slightly deflected (scattered) in the sample pass through the phase plate, bypassing the phase ring and do not undergo an additional phase shift. Taking into account the phase shift in the material of the specimen the total phase difference between undeflected and deflected rays is close to 0 or λ/2, and as a result of the light interference in the image plane of the specimen they significantly strengthen or weaken each other, resulting in a contrast image of the specimen structure. The deflected rays have much smaller amplitude as compared with the undeflected ones, therefore the weakening of the main beam in the phase ring, bringing together the amplitudes, also result in greater contrast of the image.

    The resultant image is called a phase contrast image.

    The method makes it possible to distinguish small elements of the structure, almost invisible in the bright-field observation. Transparent particles, which are rather big in size, scatter light rays at such small angles that the rays together with the undeflected ones pass through the phase ring. For such particles a phase contrast effect occurs only near their outlines, where the strong scattering appears.


∙   MI - 2 (T)