Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 13

of extent of phase shifts and diffraction. To cope with this range, different objectives can be
used to find an optimal balance of the amount of surround light that needs to be blocked by
the phase plate (based on the extent of diffraction by the sample). But these are generally
different complete objectives so the cost of acquiring this variability is fairly significant.
1.4 Experimental Uses
Phase contrast is one of the most popular, versatile, and widely used transmitted light
contrast techniques used today. It is difficult to represent adequately the breadth of uses in
the biomedical research community, but this will be attempted by presenting some general
classes of examples.
1.4.1 Cell Culture Models
Cell culture models are a very common research tool, and phase contrast is ideally suited to
the routine observation of these cells. Simple and relatively inexpensive inverted microscopes
with low magnification or higher magnification long working distance dry lenses with phase
contrast optics are situated in nearly all tissue culture facilities. They provide a quick and
effective means of assessing the health and density of the culture cells in the plastic culture
dishes and flasks used. The key advantage of phase microscopy for this purpose (as well as
simplicity and low cost) is the ability to image through relatively thick optically imperfect
plastic. The plastic used in culture vessels tends to present relatively little absorption and
local differential phase shift of the light but significant interaction (principally birefringence)
with polarized light meaning techniques such as DIC are not achieved with good results
without specialized versions of these optical contrast methods. See Chapter 3 for excellent
description of how phase can be used in live cell time-lapse studies.
1.4.2 Image Analysis
Threshold-based image analysis routines allow quantification of a broad range of features of
samples. Examples include automated counting of cells, measurement of areas, and tracking
the movement of live cells in a time lapse. Thresholding involves physically identifying an
object by a difference in intensity between the object of interest and background. Phase
contrast has two advantages for certain uses of this. The first advantage is that it provides
large contrast—the difference in intensity between the cell edge and the background—
making identification easier and more robust than with the use of brightfield images.
Anybody who has ever attempted any image analysis with biological samples using
threshold-based identification will know that with more contrast available, the easier and
more accurately one can pick apart the objects of interest and the task can range
enormously in difficulty with, among other factors, the level of contrast available.
Typically, transmitted light microscopy does not provide as high a level of contrast as does