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3025 Microscope
Phase Contrast
– Turret System
SUPPLEMENTAL INSTRUCTIONS
PHASE CONTRAST MICROSCOPY
The normal microscopic object is seen because it has regions of varying density. In normal brightfield illumination a
completely transparent specimen is very difficult to observe in detail because all areas of the specimen are equally dense.
Darkfield illumination displays border effects in completely transparent specimens due to edge scattering and diffraction of
light. Polarized light is useful when transparent specimens have directional or crystalline properties.
Phase contrast microscopy is a type of illumination system to observe transparent media. This form of illumination is
utilized extensively in the study of transparent living cells without the need for staining or fixing while being able to obtain
good image contrast. The light from phase contr
ast illumination arrives at the user’s eyes at ½ the normal wavelength.
This light altering system produces a visible image of an otherwise invisible, transparent specimen.
The optical light path necessary for phase contrast is shown in Figure 1. A clear annulus in the focal plane of the
condenser is imaged at infinity by the condenser and then re-imaged by the objective in its rear focal plane. The
undiffracted light passes through this image. It is reduced in intensity and given a one-quarter wave phase shift by means
of an annular phase pattern in the rear focal plane of the objective. These two changes in the undiffracted portion of the
beam simulate the phase and intensity distribution which would be present in the objective focal plane if the specimen had
density variations rather than refractive index variations. As a result, the image formed by the beam interfering with the
diffracted beam simulates that of a specimen having density variations.
IMAGE FORMATION BY PHASE CONTRAST
An annular aperture in the diaphragm placed in the focal plane of the substage condenser controls the illumination of the
specimen. The aperture is imaged by the condenser and objective at the rear focal plane or at the exit pupil of the
objective. A phase shifting element, or phase plate, is placed in the image plane. Light passing through the phase altering
pattern acquires a ¼ wave length advance over that diffracted by the object structure and passes through that region of
the phase plate not covered by the altering pattern. The resultant interference effects of the two portions of light form the
final image. Altered phase relations in the illumination rays, induced by otherwise invisible elements in the specimen, are
translated into brightness differences by the phase altering plate.
