Phase Imaging in Plant Cells and Tissues - Biomedical Optical Phase Microscopy and Nanoscopy - page 58

10 µ m
Figure 4.6
An illustration of the process of image production in a DIC microscope.
Nomarski in 1955 [14] developed this methodology using polarized light but versions using
unpolarized light also existed in the Interphako microscope. However, the highest apertures
and the best focal depths were given by Nomarski's methodology and it can also be
extended to imaging in three dimensions by transfer of specimens in the visual field from
one end to the other of the field of view thus creating two distinct illumination angles
yielding a combined 3D effect as in Figure 4.6 (Sarafis, 1984 with the agreement also of
Nomarski) [15] .
It is possible to make a lower resolution image by DIC from data such as shown in the
bright-field image in ( Figure 4.7 ) on phase contrast imaging using software but they are not
as good as direct DIC microscopy.
4.6 Hoffmann Modulation Contrast
HMC is a method that makes phase gradients to be displayed at different levels of intensity
relative to the background, such that the transparent object can be visualized on a
microscope [1] . It can be retrofitted to any microscope and gives relief contrast, high
resolving power in the images, and a small depth of focus [16] .
The HMC microscope works by placing a modulator at the back focal plane of the
objective. This modulator consists of three regions that have different transmission levels
(usually 1%, 15%, and 100%). Also, a slit has to be placed at the front focal plane of the
substage condenser and calibrated to allow the incident light passing through the region of
the modulator that has 15% transmission level, such that the background of the image