Differential Interference Contrast Microscopy (DIC) - Biomedical Optical Phase Microscopy and Nanoscopy - page 29

Figure 2.5

DIC images of 7 μ m diameter glass rod captured in white light with zero bias (A), 100 nm bias (B),

and 550 nm bias (C).

the optical path difference introduced by the rod side is subtracted by same amount of the
bias (see formula (2.2) ), and equals 100 nm. Figure 2.5C illustrates a case when bias is
550 nm. A full-wave plate U-TP530 was inserted in the optical path. Because the optical path
difference is about one wavelength, the image shows Newton's interference colors. Then, the
Michel-Levy chart is used to estimate the optical path difference [9,39] . The blue and orange
colors of the rod sides correspond to 650 and 450 nm of optical path difference, respectively.
After subtraction of the bias, the rod sides are shown to introduce about 100 nm of optical
path difference. According to Table 2.1 , the shear distance between the two interfering beams
is 240 nm. Then, the optical path difference gradient of the rod side is 0.4 nm/nm. Hence,
conventional DIC technique with calibrated bias can provide quantitative results.

2.4 A Quantitative OI(Orientation-Independent)-DIC Microscope with
Fast Modulation of Bias and Shear Direction

OI-DIC technique records phase gradients within microscopic specimens quantitatively and
independently of their orientation [6,20,21] . Unlike the other forms of phase and
interference microscopes, this approach does not require a narrow illuminating cone. The
new system is probably one of the few, if not the one and only, microscope system, which
allows the generation of interference microscope images at truly high NAs.

A schematic of beam-shearing assembly with high speed switching shear direction and
changing bias without any mechanical movement is shown in Figure 2.6 (center). The
assembly consists of a linear polarizer P, first variable retarder (liquid crystal cell) LC1
with the principal axis orientated at 0 , a pair of DIC prisms (Nomarski or Wollaston)
DIC1 and DIC2 with orthogonal shear directions, and second liquid crystal cell LC2
between the prisms. The second liquid crystal cell has the principal axis orientated at 45 .