Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 10

difference in the phase plates, directions of phase shift of the S-waves, and examples of
images of the same mammalian cell produced by positive and negative phase contrast.
1.2.4 Halo Artifacts
Phase contrast images have a ring around the larger objects—for positive phase contrast this
is a ring of bright light that surrounds the object, for negative phase contrast it is a dark
ring that is between object and background. What causes the phase halo and why is it only
seen around the outer edge of large objects? The halo is caused by light which is diffracted
and phase shifted but not diffracted by a magnitude or direction that causes it to pass
through the central part of the phase plate. The phase ring is normally made to be slightly
larger than the conjugate size of the annulus so it efficiently accepts the surround light after
the slight aberrations in the optics act to expand the size of the illumination ring. This
makes the system slightly more susceptible to halo than would be expected. Large objects
with lower spatial frequencies diffract the light at a lesser angle than finer scale structures
represented by high spatial frequencies, so the light that comes from large objects accounts
for the majority of this phase shifted light that passes “incorrectly” through the ring of the
phase plate. Because this light is phase shifted by the phase plate in the wrong direction
relative to the surround wave, no means of destructive interference are produced for this
light and so a high intensity region is produced. The halo has both good and bad properties:
good because it provides a strong intensity change around the edge of the cell, highlighting
this region; bad because the spatial accuracy of the definition of the edge is reduced by the
overwhelming contrast and because the relationship between intensity and optical path
length differences is lost in this part of the image.
Shade-off is a related artifact in large homogeneous objects. The center of such objects
appears brighter in positive phase contrast because this light is little deviated and so, just as
in the halo, not acted upon by the phase contrast system to produce attenuation.
1.3 How to Acquire Phase Contrast Images
1.3.1 What Components Are in a Phase Contrast System?
Good news: only two extra parts are required over brightfield and the general layout
remains compatible with other standard transmitted and epi-illumination imaging techniques
and modalities. As described earlier, phase contrast is based on a ring-shaped illumination
and a matching phase plate, most commonly in the objective itself. The illumination can be
achieved by introducing a mask in the aperture plane in the condenser. These are simple
metal disks with different sized rings; larger rings are used for higher power lenses. Most
commonly manufacturers refer to phase condenser elements with Arabic numbers such as
phase 1, 2, and 3 (Roman numerals are generally used for DIC).