Phase Contrast Microscopy

Phase contrast microscopy is used to image transparent media. It works by

converting the phase change in light passing through transparent media to brightness
changes in the image. We can’t see phase shifts visibly, but it can be inferred by the change
in the brightness. Photographic equipment and human eyes are only able to see differences
in the amplitude of light that is transmitted or reflected through scattering or absorption in
a material, but phase changes also carry lots of important information.

Working principle

The basic principle to make the phase change visible is the separated the
background light from the scattered light and manipulate them differently.

Figure 1 – Schematic of a phase contrast microscope. From [1]

The illuminating light (green) passes through a condenser annulus and is focused into the
specimen by the condenser. Some light is scattered by the specimen (yellow) and some
passes through unscattered (red). The scattered light has a difference in optical path length
(OPL) compared to unscattered light, given by:

𝑂𝑃𝐿 = 𝑛 𝑥 𝑡

where n is the refractive infect of the object and t is the thickness of the object.

Since in general the specimen has a higher refractive index than the surrounding air, light
that is scattered through the specimen will have gained phase in radians given by:
 𝛿 = 2𝜋∆/𝜆
where ∆ is the optical path difference between the scattered and unscattered light and 𝜆 is
the wavelength of light.
In phase contrast microscopy, the scattered and unscattered light typically has a 𝜋/2 phase
shift, although there is a phase shift, the transmitted light have the same intensity, so the
resulting image has low contrast.

This contrast is increased in two ways:

-by generating constructive interference between the scattered and background light rays in
the regions of the field of view that contain the specimen.
-by reducing the amount of background light that reaches the image plane.

To generate constructive interference between the scattered and background light rays, a
quarter wave plate is used to shift the background light - 𝜋/2, so it is at the same phase as
the scattered light. This causes the background and scattered light from the sample to
constructively interfere, therefore increasing the brightness of the scattered areas
compared to the parts that don’t contain any of the specimen. This is called negative phase
contrast. Positive phase contrast can also be achieved by phase shifting the background
light by 90 degrees again, thereby making it out of phase by a factor of 𝜋. If this method is
used, the scattered light with be subtracted from the background light to form an image
with a dark foreground and light background. The differences are shown below.

Figure 2 – Comparison of positive and negative phase contrast images. (a),(c),(e) and
(b),(d),(f) showing positive and negative phase contrast images respectively. Edited from [2]

To reduce the amount of background light, a neutral density filter is used to dim around 70-
90% of the total light. This serves to maximise the amount of scattered light that is
measured while minimising the amount of background light.
Interpretation of images

The optical path difference between the specimen should be carefully considered to
make sure that the light and dark parts of the image are understood correctly. Since the
optical path difference and therefore the phase shift is due to the thickness of the
specimen, the relative thickness of all parts must be well known. To increase the image
contrast the surrounding medium of the specimen can be changed to a lower or higher
refractive index material, in a method known as immersion refractometry.

Two main drawbacks of phase contrast imaging are the occurrence of halos and
shade off contrast patterns where the OPD and observed intensity don’t directly correspond
to each other. These effects can be seen below.

Figure 3 – Halos and shade off contrast patterns. From [3].

The halo effect can also be significantly reduced by utilizing specially designed phase
objectives that contain a small ring of neutral density material surrounding the central
phase ring material near the objective rear aperture. With the apodized optics, contrast is
reversed due to the large amplitude of diffracted light relative to that of the direct light
passing through the specimen.

Shade-off is another very common optical artifact in phase contrast microscopy, and is often
most easily observed in large, extended phase specimens. It would normally be expected
that the image of a large phase specimen having a constant optical path length across the
diameter would appear uniformly dark or light in the microscope. Unfortunately, the
intensity of images produced by a phase contrast microscope does not always bear a simple
linear relationship to the optical path difference produced by the specimen. Other factors,
such as absorption at the phase plate and the amount of phase retardation or
advancement, as well as the relative overlap size of the phase plate and condenser annulus
also play a critical role.

Halo and shade-off artifacts depend on both the geometrical and optical properties of the
phase plate and the specimen being examined. In particular, the width and transmittance of
the phase plate material play a critical role in controlling these effects (the phase plate
width is typically about one-tenth the total aperture area of the objective). Wider phase
plates having reduced transmittance tend to produce higher intensity halos and shade-off,
whereas the ring diameter has a smaller influence on these effects. For a particular phase
objective (either positive or negative), the optical path difference and specimen size, shape,
and structure have significant influence on the severity of halo and shade-off effects.

[1] https://en.wikipedia.org/wiki/Phase-contrast_microscopy
[2] http://www.bwoptics.com/newsend2.asp?id=3
[3] https://www.microscopyu.com/techniques/phase-contrast/introduction-to-phase-
contrast-microscopy