Differential Interference Contrast Microscopy (DIC) - Biomedical Optical Phase Microscopy and Nanoscopy - page 25

The above data are shown for the green light ( λ 5 532 nm). The shear angle was about 4%

less for the red light ( λ 5 641 nm).

We have measured parameters of the Olympus DIC prism U-DICTH, which was
manufactured in previous years. Its shear angle is the same as the prism U-DICTHR. Mehta
and Sheppard [23] found the angular shear of 74 rad for the U-DICTS prism at wavelength

550 nm. This shear corresponds exactly to our observations for the U-DICT prism. Our
other results indicate that shear angles of Nikon 60xI and Zeiss PA63x/1.40III DIC sliders
are 76 and 71

μ

μ rad, respectively.

2.3 Bias Optimization

In polarization microscopy, the Br¨ce-K¨hler compensator, an azimuthally
rotatable birefringent plate with bias retardance up to /10, allows investigation of a

specimen with small birefringence more precisely than the Senarmont compensator, which
has a retardance of

λ

/20
Br¨ce-K¨hler compensator, could detect a 0.028 nm retardation. In order to achieve high
sensitivity with the LC-PolScope (a commercially available polarization microscope), we
apply alternate bias retardance

λ /4 [7,26] . For example, Swann and Mitchison [27] , using the λ

/30 [28,29] . The measured noise level using the five-frame
algorithm was 0.036 nm. The bias

λ

/5, which was used for studying a sample with large
retardance, produces much higher noise.

λ

In conventional DIC microscopy, the situation is similar. Near extinction, the image field
becomes dark gray and yields very high sensitivity, bringing out image regions with
minute phase differences due to, for example, extremely shallow depressions or elevations
or from isolated subresolution filaments. Use of bias less than 90 is advantageous in
video-enhanced-DIC (VE-DIC) microscopy [30,31] . Holzwarth [12] studied a photon noise
versus bias in PM-DIC microscopy. He showed theoretically and confirmed experimentally
that the signal-to-noise ratio peaks when the bias equals the sample optical path difference.

The benefit of the optimized bias can be proved mathematically by using the following
example. Let us consider a simplified sample with a binary gradient magnitude distribution
such that half of the sample has a gradient magnitude of 1 γ and the other half has a phase

difference of 2 γ (see Eq. (2.2) ). The shear and gradient directions of the sample are

θ 5 0 ). We also take into account the depolarized light by using an extinction

parallel (

I ~ Þ: ratio ξ 5 ð I c =

We expect the best results to be achieved when the DIC images have the highest contrast C :

I max 2 I min
I max 1 I min

C 5 (2.13)

where I max and I min are the maximal and minimal intensities in the image.