Biomedical Engineering Reference
In-Depth Information
resolved if the microscope objective captures the zeroth order of the light
(undeviated light) and at least the fi rst order (deviated or diffracted light), or
any two orders. Unstained specimens that do not absorb light are called phase
objects or specimens because they alter the phase of the light diffracted by
the specimen without a change in its amplitude or intensity. These specimens
retard the diffracted light approximately one-quarter of a wavelength as com-
pared to the undeviated light passing through or around the specimen unaf-
fected. The human eye, as mentioned before, can only detect variation in the
colors of the visible spectrum or differing levels of light intensity due to wave
amplitude differences. In essence, the eye cannot detect the phase differences
described above. Phase specimens yield images in the microscope almost
totally lacking in contrast which make specimen details almost invisible.
By using phase contrast microscopy, the analyst can obtain images of
unstained phase specimens having contrast levels similar to light absorbing
or amplitude specimens. The human eye perceives contrast at acceptable
levels in amplitude specimens when the diffracted and undeviated light rays
are out of phase by one-half of a wavelength. In phase contrast microscopy
the velocity of the undeviated light is speeded up by one-quarter of a wave-
length so that the difference in wavelength between the undeviated and dif-
fracted light for a phase specimen would now be one-half of a wavelength.
As a result, the undeviated and diffracted light arriving at the image plane
of the eyepiece would now produce destructive interference. The increased
phase shift results in the details of the image produced appearing darker
against a lighter background. This is called positive phase contrast. An alter-
native phase contrast operating mode produces images with bright details on
a darker background. This is called negative phase contrast. Positive phase
contrast is the most commonly used imaging mode with modern research
grade phase contrast microscopes.
Phase contrast is produced by separating the undeviated zeroth order
light from the diffracted at the back focal plane of the microscope objective.
This is accomplished by placing a ring annulus, a circular plate containing
a transparent glass circle with an opaque or non-transparent central stop,
in the position directly under the lower lens of the substage condenser at
its front focal plane. A hollow cone of light is produced by the annulus;
it travels through the specimen undeviated and arrives at the back focal
plane of the objective as a ring of light. The less intense light diffracted by
the specimen is spread evenly over the back focal plane of the objective.
A phase plate containing a ring-shaped phase shifter is attached to the
back focal plane of the objective to speed up the velocity of the undeviated
zeroth order light. The narrow circular area of the phase ring is optically
thinner than the rest of the plate. This condition allows the undeviated light
from the specimen passing through the phase ring to travel a shorter optical
path than the light diffracted by the specimen. When the undeviated and
