Page 118 - Fundamentals of Light Microscopy and Electronic Imaging

PHASE CONTRAST MICROSCOPY 101

deviated or diffracted wave (D wave) that becomes scattered in many directions. Typi-
cally, only a minority of incident waves are diffracted by cellular objects. Both S and D
waves are collected by the objective lens and focused in the image plane at the site cor-
responding to the image of the particle, where they undergo interference and generate a
resultant particle wave (P wave). The relationship among waves is thus described as
P S D. Detection of the object image depends on the intensities, and hence on the
amplitudes, of the P and S waves. Only when the amplitudes of the P and S waves are
significantly different in the image plane can we see the object in the microscope. Before
beginning our explanation of the interference mechanism, we should note our earlier
discussion of the coherence of light waves in the myriad small beams (wave bundles)
illuminating the specimen (Chapter 5). This condition is of great practical importance
for phase contrast microscopy, because image formation through constructive and
destructive interference requires coherent illumination such that: (1) A definite phase
relationship exists between the S and D waves, and (2) the phase relationship must be
preserved between the object and the image.

DEPICTION OF WAVE INTERACTIONS WITH SINE WAVE
AND VECTOR DIAGRAMS
With this general scheme in mind, let us now examine Figure 7-5a, which shows the S,
D, and P waves as sine waves of a given wavelength in the region of the object in the
image plane. The S and P waves, whose relative intensities determine the visual con-
trast, are shown as solid lines, whereas the D wave (never directly observed) is shown as
a dashed line. The amplitude of each wave represents the sum of the E vectors of the
component waves. The D wave is lower in amplitude than the S wave, because there are
fewer D-wave photons than there are S-wave photons at the image point. Notice that the
D wave is retarded in phase by /4 relative to the S wave due to its interaction with the
object particle. The P wave resulting from interference between the D and S waves is
retarded relative to the S wave by only a small amount ( /20) and has an amplitude
similar to that of the S wave. Since the S and P waves have close to the same amplitude

For a physicist interested in optics it was not a great step to change over from this
subject to the microscope. Remember that in Ernst Abbe’s remarkable theory of the
microscope image the transparent object under the microscope is compared with a grating.
Abbe examined gratings made of alternating opaque and transparent strips (amplitude
gratings). Gratings made of transparent alternating thick and thin strips (phase gratings)
produce diffraction spots that show a phase difference of 90°. For a phase object, my phase
strip in the focal plane of the microscope objective brought the direct image of the light
source into phase with the diffracted images, making the whole comparable to the images
caused by an amplitude object. Therefore the image in the eyepiece appears as that of an
absorbing object—that is, with black and white contrast, just as if the object has been
stained.
On looking back on these events I am impressed by the great limitations of the human
mind. How quick we are to learn—that is, to imitate what others have done or thought
before—and how slow to understand—that is, to see the deeper connections. Slowest of all,
however, are we in inventing new connections or even in applying old ideas in a new field. In
my case, the really new point was the diffraction pattern of lines of the grating artifacts, the
fact that they differed in phase from the principal line, and that visualization of phases
required projection of the diffraction image on a coherent background. The full name of the
new method of microscopy might be something like “phase-strip method for observing phase
objects in good contrast.” I shortened this to “phase contrast.”