Phase contrast microscopy is an optical microscopy technique that converts phase
shifts in light passing through a transparent specimen to brightness
changes in the image. Phase shifts themselves are invisible, but become
visible when shown as brightness variations.
When light waves travel through a medium other than vacuum, interaction with the
medium causes the wave amplitude and phase to change in a manner dependent on
properties of the medium. Changes in amplitude arise from the scattering and
absorption of light, which is often wavelength dependent and may give rise
to colors. Photographic equipment and the human eye are only sensitive to
amplitude variations. Without special arrangements, phase changes are
therefore invisible. Yet, phase changes often carry important information.
Phase contrast microscopy is particularly important in biology. It
reveals many cellular structures that are not visible with a simpler bright
field microscope, as exemplified in Figure 1. These structures were made
visible to earlier microscopists by staining, but this required additional
preparation and killed the cells. The phase contrast microscope made it
possible for biologists to study living cells and how they proliferate through
cell division. After its invention in the early 1930s, phase contrast
microscopy proved to be such an advancement in microscopy, that its
inventor Frits Zernike was awarded the Nobel prize in 1953.
Working principle The basic principle to make phase
changes visible in phase contrast microscopy is to separate the
illuminating background light from the specimen scattered light, which make up
the foreground details, and to manipulate these differently.
The ring shaped illuminating light that passes the condenser annulus is focused
on the specimen by the condenser. Some of the illuminating light is scattered
by the specimen. The remaining light is unaffected by the specimen and forms the
background light. When observing an unstained biological specimen, the
scattered light is weak and typically phase shifted by -90° — relative to the
background light. This leads to the foreground and background having nearly
the same intensity, resulting in a low image contrast.
In a phase contrast microscope, the image contrast is improved in two steps.
The background light is phase shifted -90° by passing it through a phase shift
ring. This eliminates the phase difference between the background and
the scattered light, leading to an increased intensity difference between
foreground and background. To further increase contrast, the background is
dimmed by a gray filter ring. Some of the scattered light will be phase
shifted and dimmed by the rings. However, the background light is
affected to a much greater extent, which creates the phase contrast effect.
The above describes negative phase contrast. In its positive form, the
background light is instead phase shifted by +90°. The background light
will thus be 180° out of phase relative to the scattered light. This results in
that the scattered light will be subtracted from the background light in
to form an image where the foreground is darker than the background, as shown in
Figure 1. Related methods
The success of the phase contrast microscope has led to a number of
subsequent phase imaging methods. In 1952 Georges Nomarski patented what is
today known as differential interference contrast microscopy. It enhances
contrast by creating artificial shadows, as if the object is illuminated from the
side. But, to achieve this DIC microscopy uses polarized light, making
it unsuitable when the object or its container alter polarization. With the
growing use of polarizing plastic containers in cell biology, DIC
microscopy is increasingly replaced by hoffman modulation contrast microscopy,
invented by Robert Hoffman in 1975. Traditional phase contrast methods
enhance contrast optically, blending brightness and phase information in
single image. Since the introduction of the digital camera in the mid-1990s,
several new digital phase imaging methods have been developed,
collectively known as quantitative phase contrast microscopy. These methods
digitally create two separate images, an ordinary bright-field image and a
so-called phase shift image. In each image point, the phase shift image
displays the quantified phase shift induced by the object, which is
proportional to the optical thickness of the object.
See also Live cell imaging
Phase-contrast imaging Phase-contrast X-ray imaging
References External links
Optical Microscopy Primer — Phase Contrast Microscopy by Florida State
University