Under these conditions, the objective rear focal plane is conjugate to the condenser front aperture plane, so non-diffracted (zeroth order) light waves form a bright image of the condenser annulus at the rear aperture of the objective (superimposed over the image of the phase plate). Under conditions of Köhler illumination, surround light waves that do not interact with the specimen are focused as a bright ring in the rear focal plane of the objective (the diffraction plane). Phase contrast is also insensitive to polarization and birefringence effects, which is a major advantage when observing tissue culture cells growing in plastic vessels. Unlike differential interference contrast and Hoffman modulation contrast, the circular geometry of phase contrast illumination and detection enables specimen observation without orientation-dependent artifacts. When conducting phase contrast experiments utilizing a condenser with both a phase annulus and an aperture diaphragm, check to ensure that the iris diaphragm is opened wider than the periphery of the phase annulus. The condenser annulus either replaces or resides close to the adjustable iris diaphragm in the front aperture of the condenser. This concept is useful for describing the configuration, but is not strictly accurate. It should be noted that many texts describe the illumination emergent from the condenser of a phase contrast microscope as a hollow cone of light with a dark center. The microscope condenser images the annular diaphragm at infinity, while the objective produces an image at the rear focal plane (where a conjugate phase plate is positioned, as discussed below). The condenser annulus (illustrated in Figure 1) is typically constructed as an opaque flat-black (light absorbing) plate with a transparent annular ring, which is positioned in the front focal plane (aperture) of the condenser so the specimen can be illuminated by defocused, parallel light wavefronts emanating from the ring.
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