Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 8

The two waves then interfere and a high contrast image is produced at the image plane that
may be captured by CCD or CMOS camera, or viewed with extra magnification through
eyepieces. Maximum extinction will be produced by regions or objects which produce a
deviated wave that has a phase shift which when summed with the phase shift introduced
by the phase plate produce a total shift of /2 and is of intensity comparable to the

attenuated surround wave. A heterogeneous biological sample will also produce a range of
phase shifts and extents of diffraction to have a range of contrast.

λ

1.2.2 What Do You Actually See in a Phase Contrast Image?

The contrast added by exploiting phase effects within a sample clearly reveals many details
not otherwise visible. With understanding of the method of phase contrast, it is easy to
remember that darker regions of the image are not accounted for by absorption. The edge of a
culture cell is clearly defined because of the difference between the propensity for diffraction
and the phase shift in the cell and space surrounding it. But what are the biological structures
or materials that give contrast within a sample? What of the organelles inside a cell—what is
visible, what is not? With knowledge of the structure of a cell, it is clear that many familiar
structures are visible—the nucleus, the nucleolus, and the vacuoles are clearly defined and
identifiable. Other organelles are typically less clear but can be seen (e.g., mitochondria).

The simplest general explanation of the difference in contrast across a sample is the
difference in optical path length (as defined earlier, this is the product of refractive index
and thickness) at different points. Organelles and fine structures within the cell produce
small local differences in refractive indices but because the thickness of the cell is also
involved in the optical path length, the contrast distribution cannot be simply interpreted in
terms of the refractive index. Often ruffles are visible in cells (see Figure 1.1 ), and these
could be interpreted as differences in thickness of the cell with a relatively homogeneous
refractive index of the cytosol but the precise contribution of the two components of optical
path length are never known for sure.

The regions that appear darkest in a positive phase contrast image are those that present the
extent of diffraction and phase shift from optical path length difference to the surround
that produce a total phase shift offering highest extinction. Regions with a total phase
shift differing from this maximum will appear lighter. It is not always trivial to attribute
differences in intensity to particular directions of phase shift from optical path length
differences since large, say, retardations may produce the same effect as a small advance.
Indeed, thick objects with large optical path differences could produce a phase shift of more
than one wavelength.

Even in the absence of linear direct quantitative attribution between levels of contrast
observed, the magnitude of the phase shift that produces the contrast and the precise