Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 9

biological materials or subcellular structures that account for the contrast, the definition
produced by phase contrast is extremely useful.

1.2.3 Positive and Negative Phase

The principles discussed earlier were explained in terms of positive phase contrast which is
the most popular implementation in biological applications. Positive phase contrast involves
retardation by the sample, and advance of the surround wave by the phase plate to produce
λ /2 total relative phase shift for maximum extinction. A negative phase plate can be used

and the principle of contrast formation is essentially the same except the phase plate
produces a relative shift between diffracted and surround light in the opposite direction.
In negative phase contrast, the surround wave is retarded relative to the diffracted wave and
so regions of phase retardation within the sample produce positive interference and appear
brighter than the background. It is somewhat a matter of sample-specific taste whether
positive or negative phase gives better results. Generally, positive phase contrast is found to
be more clear and these objectives are more commonly available. Figure 1.7 shows the

(A) (B)

S S

D

D

P P

Figure 1.7
(A) Positive and (B) negative phase contrast.