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Phase Contrast Microscope

When a beam of light passes through a transparent object, phase difference is produced but no change in amplitude takes place. The eye can distinguish only changes in intensity but not changes in phase. In case one wishes to see a small transparent object say unstained bacteria, it is necessary to magnify it and also to convert differences in phase into differences in intensity. Zernike in 1935 introduced the concept of phase contrast.

Consider a beam of light passing through a transparent plate of varying thickness. The amplitude vector at the points A, B, C has the same magnitude but is in different directions. The intensity is the same at all points but there is difference in phase cannot be seen by the eye. However, if a constant phase difference is introduced with the help of a phase plate (represented by dotted vector), the resultant amplitudes at the points A, B and C are R₁, R₂ and R₃ respectively. Their magnitudes are different and hence the intensities are different and can be seen by the eye. Thus, by introducing the phase plate, the object slide can be seen by phase contrast with the help of the phase plate, phase changes due to varying thickness are converted to amplitudes of varying magnitude at different points.

A phase contrast microscope consists of a transparent object slide placed in between the condenser and the objective. An annular aperture is placed at the first focal plane of the condenser and the phase plate is kept at the second focal plane of the objective. The phase plate has an annular depression exactly conjugate to the annular aperture.

In the figure, the path of zero order light is shown. It is clear that the light passes through the thinner part of the phase plate and consequently its phase is advanced in comparison to the light diffracted through the thicker part of the plate. If t is the thickness of the depression and is the refractive index of the phase plate, the advance in phase of the zero order light is . For maximum efficiency, the image of the annular aperture should coincide exactly with the depression in the phase plate. The final image is formed at the first focal plane of the eyepiece.