Differential Interference Contrast Microscopy (DIC) - Biomedical Optical Phase Microscopy and Nanoscopy - page 27

high extinction in order to effectively use a small bias for studying tiny structures. The
contrast of cell walls and organelle edges with a phase difference λ /10 is 0.79 at bias λ /20

and infinity extinction, 0.72 at bias λ /20 and extinction 100, and 0.59 at bias λ /4 and both

extinctions.

In addition to reducing contrast, the low extinction decreases the dynamic range of
measurement and can cause a diffraction anomaly in the Airy pattern. The lower extinction
is a significant problem in microscopes equipped with high NA lenses. To reduce the beam
depolarization, we can use a polarization rectifier [33,34] .

Most current DIC microscopes employ one of the following methods of changing bias: (a)
lateral shift of the DIC prism using a screw, (b) the Senarmont compensator, or (c) liquid
crystal variable retarder. In principle, it is also possible to use other means for the bias
adjustment. For example, one can employ the Babinet-Soleil compensator [26] , the
Ehringhaus compensator [26,35] , the Berek compensator [26,36] , or its analogue made of
quartz [37] , electro-optic, or piezo-optical modulators [38] . All of these methods could be
readily calibrated for the quantitative bias variation.

The total optical path difference (bias) between two interfering beams is created by
combination of two DIC prisms (see Figure 2.1 ). Each of the prisms introduces bias, which
is not uniform and has a gradient. However, the prisms are oriented such that they mutually
compensate the bias gradient. Thus, the total bias distribution becomes even across the
objective back focal plane. The bias can be changed by a lateral shifting of one of the DIC
prisms along the bias gradient direction. As it was shown in Section 2.1 ( formula (2.4) ), the
unitless bias gradient equals the shear angle (in radians). The linear equation for the current
bias Γ ( x ) is written:

Γð x Þ 5 Γ 0 1 εð x 2 x 0 Þ (2.16)

where x and x 0 are the current and initial positions of the prism and Γ 0 is the initial bias.

The shear angle ε can be found in Table 2.1 or it can be measured, as described in the

previous section.

Usually, the DIC prism is shifted by a translation screw. Then, the bias variation is
determined using the pitch of a screw thread p and revolution number R in the following
way:

Γð R Þ 5 Γ 0 1 ε pR (2.17)

For example, the Olympus high-resolution DIC prism U-DICTHR has a translation screw
with pitch of 2.5 mm. The screw allows a maximum of five rotations. According to
Table 2.1 , the prism shear angle is 40

Γ 5 100 nm
per 360 turn of the screw and the total range of the bias change Γ tot 5 500 nm is
calculated. The General Olympus DIC prism U-DICT has 3 mm screw pitch and maximum

μ

rad. Using Eq. (2.17) , the bias variation