Phase contrast microscopy

Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image



4. Primary image plane
3. Phase plate (phase ring)
2. Object plane
1. Condenser annulus (round hole)

The light emanating from the small aperture 'S' is captured by the condenser and emerges as light with only parallel wavefronts from the condenser. When these parallel wave fronts hit the phase object 'O', some of this light is diffracted (and/or refracted) while moving through the specimen.

Newly generated spherical wave fronts that are retarded by 90° (λ/4) emanate from 'O' (see the purple area that contains now "unperturbed" plane waves and spherical wave fronts). It is important to note that there are now two types of waves, the surround wave or S-wave (pink orange) and the diffracted wave or D-wave (blue), which have a relative phase-shift of 90° (λ/4).

The objective focuses the D-wave inside the primary image plane ('4'), while it focuses the S-wave inside the back focal plane ('3'). The phase plate 'P' reduces the amplitude of all light rays traveling through the phase annulus (mainly S-waves) by 70 to 90% and advances the phase by yet another 90° (λ/4). It leaves most of the D-waves "untouched". Hence the recombination of these two waves (D + S) in the primary image plane (labeled '4') results in a significant amplitude change at all locations where there is a now destructive interference due to a 180° (λ/2) phase shifted D-wave.

Phase contrast amplifies edges without needing stains. But might have halos.