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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.Characteristics
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, “Video Microscopy?(2001),
Kyoritsu Shuppan |
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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. |
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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
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