Differential Interference Contrast Microscopy (DIC) - Biomedical Optical Phase Microscopy and Nanoscopy - page 33

The gradient magnitude represents the increment of the optical path difference, which is in
nanometers, along the lateral coordinate, which is also in nanometers. Thus, the gradient
magnitude is unitless. The shear amount d can be measured as it is described in Section 2.1
or found in Table 2.1 .

Note that the algorithm considered earlier employs ratios between intensities of light that
have interacted with the specimen. Therefore, it suppresses contributions of absorption by
the specimen or from nonuniformity of illumination, which can otherwise deteriorate a DIC
image.

Also, after computing the optical path gradient distribution, enhanced regular DIC images
I enh ( x , y ) can be restored with any shear direction σ , different bias Γ , and another shear

amount d using the next formula:

π
λ Γ 1 dγðx; yÞ

I enh ðx; yÞ 5 sin 2 ½ cos ðθðx; yÞ 2 σÞ (2.24)

The enhanced image provides a calculated image for any desired shear direction and bias
without the requirement to directly collect an image for that shear direction and bias.
Moreover, the enhanced image will have less noise than a regular DIC image, and it
suppresses deterioration of the image due to specimen absorption and illumination
nonuniformity.

Optical path difference shows the dry mass distribution of a specimen and can be obtained
by computing a line integral [6,40] . Also, other techniques for phase computation can be
used, for instance, iterative computation [41] , noniterative Fourier phase integration [15] ,or
nonlinear optimization with hierarchical representation [42] . Biggs has developed an
iterative deconvolution approach for computation of phase images, based on the same
principles as deconvolution techniques normally used to remove out-of-focus haze [43 45] .

In principle, a regular research grade microscope equipped with DIC optics can be modified
for obtaining OI-DIC images. The setup was implemented on an upright microscope
Olympus BX-61 (Olympus, Center Valley, PA, http://www.olympus.com ) . We built two
custom beam-shearing DIC assemblies, one placed in the illumination path and the another
in the imaging path ( Figure 2.7 ). Using MATLAB (The MathWorks Inc., Natick, MA,
http://www.mathworks.com/ ) , we developed software for setup control and image
processing.

Figure 2.8 shows an example of the phase OI-DIC image of a live crane fly spermatocyte
during metaphase of meiosis I taken with an Olympus UPlanFl 100 3 /1.30 oil immersion
objective lens and 546/30 nm interference filter. Image size is 68 m. Here, the

image brightness is linearly proportional to the phase (refractive index) distribution. The
image acquisition and processing took about 1 s each. The three autosomal bivalent

μ m 3 68 μ