Biomedical Engineering Reference
In-Depth Information
CHAPTER 1
Phase Contrast Microscopy
Sam A. Johnson
Light Microscopy Core Facility, Duke University and Duke University Medical Center, Durham, NC
Editor: Lisa L. Satterwhite
1.1 Introduction
Since the mid-1600s microscopes of increasing sophistication have opened a world invisible
to the naked eye and are now ubiquitous and essential biomedical research tools.
Magnification allowed observation of some objects and structures that were previously
unknown and the capture of details impossible without the use of optics. This alone was
transformative and opened entire fields of study.
But magnification is nothing without contrast—defined as the difference in intensity
between two points divided by their average intensity—and many samples of considerable
biological interest provide very limited contrast in homogeneous brightfield transmitted
light microscopy. Phase contrast provides a means of extracting additional contrast from a
variety of samples which provide little absorption-based contrast. Contrast can also be
added by preparation and manipulation of the sample but this often involves fixing the
sample, which can confer a considerable disadvantage in the study of biology and the
dynamic processes it involves. Therefore, a method of observation of intact, live, and
unlabeled samples is key.
Figure 1.1
shows the difference between a brightfield image and a phase contrast image of
a typical thin transparent biological sample, a flat mammalian cell.
As seen in
Fig. 1.1
, phase contrast [1
5] provides additional clarity and detail in live
biological samples: more contrast is provided and finer structures can be visualized. Today,
these abilities make it an indispensable tool for many who are interested in imaging diverse
samples such as cultured cells, bacteria, thin tissue samples, and yeast. The significance and
advance that this method of contrast formation provides merited a Nobel Prize in 1953 to
Zernike for its development in the 1930s. Today, phase contrast is a very common feature
of thousands of microscopes worldwide and can be considered an absolutely core technique
