One of the key factors in Differential Interference Contrast (DIC) microscopy is "shear amount", which helps determine resolution and contrast. Shear amount refers to the minute difference in the distance within the specimen that two paths of light are made to travel by interference. The light paths are divided by Wollaston or Nomarski prisms to provide the interference patterns.

Increasing the shear amount provides greater contrast, but at the cost of resolution. When using a normal ocular lens to observe, it is common practice that the shear amount should be approximately one half that of the resolution of the objective.

Nikon uses a short-shear prism to enable imaging of extremely fine structures. Two short-shear prisms are offered, the CFI Plan Apo 60X oil prism and the CFI Plan Apo 100X oil prism.

The Plan Apo 60X oil has a shear amount of 0.04 mm and a 1.40 N.A., which is approximately 1/5 of its resolution. Normally this would render insufficient contrast to make details visible to the naked eye, but the VEC (Video Enhanced Contrast) method of electrical signal processing transforms the signal into a high contrast image.

1.Characteristicsimage

1. Enabling visualization of fine structures less than 0.2 microns across

With a DIC microscope, its maximum obtainable contrast (R) is calculated by R = 2 x S. (S is shear amount).

Decreasing shear amount provides greater resolution, but contrast suffers. This problem can be solved using bright optics with a high EF and VEC. This system allows visualization of extremely fine structures of living cells, which heretofore had been virtually impossible to observe.

D denotes the phase contrast arising from the differing refraction indexes between the specimen and its surroundings as the plane wave passes through it.

Specimen: MDCK cell

Microscope: Nikon TE300 Inverted Microscope Plan Apo 60X H/1.4

CCD camera: C2400-75w/ARGUS20

Image courtesy of Shuichi Obata, Ph.D., Department of Anatomy, Yokohama City University School of Medicine (including specimen in Figure 2)

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In order to verify the 0.2 micron resolution, electron beam lithography was used to create an assay specimen from the cross section of 1-2 (approximating a living specimen), the image of which is shown in 1-3.

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References

1) T. Otaki, T.Kawahito, US Patent 5,572,359

2) S. Inoue, K. Spring Video Microscopy (1997), Plenum Press New York

3) S. Inoue, K. Spring; translated by Susumu Terakawa, Koji Ichie, and Akira Watanabe, 밮ideo Microscopy?(2001), Kyoritsu Shuppan

2. Imaging thick specimens

Since the phase contrast for specimens with a thick end section is large, ordinary DIC techniques lead to a prominent halo effect, which inhibits visualization of fine structures (Sample image 2-1). This halo effect is reduced with high resolution DIC using short shear amount, allowing fine structures to be easily visualized, even thick specimens such as mitotic phase cells (Sample image 2-2).

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3. Extremely thin optical sections can be imaged

High aperture objectives give an extremely shallow depth of field, allowing thin optical sections to be imaged and minute processes (especially in thick specimens) to be observed effectively.

2. Related Products

TE2000U Inverted Microscope DIC Set

High Resolution DIC Attachment (high resolution system condenser oil/DIC prism/objective cassette)

ORCA Series High-sensitivity cooled CCD Camera

AQUA COSMOS Image Processing System