Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 12

Aligned Misaligned

Figure 1.8
Phase ring alignment and an example of an image produced with a misaligned phase ring.

The phase ring can be aligned by viewing the back focal plane of the objective where the
phase ring is present, and the annulus giving ring illumination is conjugate. This can be
most easily viewed by removing one of the eyepieces. Magnification can be provided by
using a Bertrand telescope but it is not impossible to align without this tool, especially for
low power lenses. Removing the sample may make this alignment easier so the diffracted
rays are not present to lessen the definition of the illumination ring. The sample was present
when the images of the back focal plane of the objective were captured for Figure 1.8 . The
diffracted rays can be seen across the complementary region, but the illumination ring is
still visible in the presence of these waves for this sample.

The condenser will have at least two screws giving fine adjustment of the position of the
ring to allow superimposition. It is important to remember that adjustment screws in the
condenser are not the same as those providing centration adjustments for K¨hler alignment.
Often once a ring is aligned for an objective, it tends to be fairly stable and may not need
adjusting with every use but the alignment is very quick to test to make sure it is correct.
The phase plate in the objective is virtually always fixed and nonadjustable.

1.3.4 Adjustability

Phase contrast requires fewer setup steps than many other contrast methods for transmitted
light imaging. In most cases for a particular objective, once it is properly set up, there
essentially are not any adjustments to make. Different samples present a substantial range