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.

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Background

The technique was invented by Frits Zernike in the 1930s for which he received the Nobel prize in physics in 1953. Phase-contrast microscopy is a mode available on most advanced light microscopes and is most commonly used to provide contrast of transparent specimens such as living cells or small organisms.

Explanation

A practical implementation of phase-contrast illumination consists of a phase ring (located in a conjugated aperture plane somewhere behind the front lens element of the objective) and a matching annular ring, which is located in the primary aperture plane (location of the condenser's aperture).

Two selected light rays, which are emitted from one point inside the lamp's filament, get focused by the field lens exactly inside the opening of the condenser annular ring. Since this location is precisely in the front focal plane of the condenser, the two light rays are then refracted in such way that they exit the condenser as parallel rays. Assuming that the two rays in question are neither refracted nor diffracted in the specimen plane (location of microscope slide), they enter the objective as parallel rays. Since all parallel rays are focused in the back focal plane of the objective, the back focal plane is a conjugated aperture plane to the condenser's front focal plane (also location of the condenser annulus). To complete the phase setup, a phase plate is positioned inside the back focal plane in such a way that it lines up nicely with the condenser annulus.

Only through correctly centering the two elements, phase contrast illumination can be established. A phase centering telescope that temporarily replaces one of the oculars is used to center the annular ring with the ring of the phase plate.

Technical Details

  To understand how phase contrast illumination works, we study two wave fronts (see the figure to the right). This figure simplifies a few things. First, the condenser annulus is just a small aperture located in the center (see the plane labeled '1') and the phase plate is also just covering a small aperture (located in the plane labeled '3'). Second, the optical system is greatly simplified by showing only two single lenses to represent all optical elements.

The plane labeled '1' is the front focal plane of the condenser. 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 plane waves (parallel wave fronts) hit the phase object 'O' (located in the object plane labeled '2'), some of this light is diffracted (and/or refracted) while moving through the specimen. Assuming that the specimen does not significantly alter the amplitudes of the incoming wavefronts but mainly changes phase relations with respect to the "unperturbed" wavefronts, 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 and the diffracted wave or D-wave, which have a relative phase-shift of 90° (λ/4). - The objective focuses the D-wave inside the primary image plane (labeled '4'), while it focuses the S-wave inside the back focal plane (labeled '3'). The location of the phase plate 'P' has now a profound impact on the S-wave while leaving most of the D-wave "unharmed". In what is known as positive phase contrast optics, 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). However, the phase plate 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. The net phase shift of 180° (λ/2) results directly from the 90° (λ/4) retardation of the D-wave due to the phase object and the 90° (λ/4) phase advancement of the S-wave due to the phase plate. Without the phase plate, there would be no significant destructive interference that greatly enhances contrast. With phase contrast illumination "invisible" phase variations are hence translated into visible amplitude variations. The destructive interference is illustrated in the figure to the right. Blue and orange indicate D-wave and S-wave, respectively. The resulting wave (D + S), indicated by yellow, has a reduced amplitude.  

See also

• Phase contrast microscope
• Microscopy
• Microscope
• Phase-contrast imaging
• Differential interference contrast microscopy

References

• Maksymilian Pluta, Advanced Light Microscopy, Vol 2, Specialized Methods, Elsevier and PWN-Polish Scientific Publishers (1989)

• Douglas B. Murphy, Fundamentals of Light Microscopy and Electronic Imaging, John Wiley & Sons (2001)