Biomedical Engineering Reference
In-Depth Information
interpreted as a true three-dimensional representation of the surface geom-
etry. One drawback with this technique is the limited depth of fi eld at higher
magnifi cations.
Optical pathway in refl ected light DIC microscopy
Light from an Epi or refl ected light illuminator passes through a polar-
izer to produce linearly polarized light that is refl ected down towards the
specimen surface by a half-mirror. On its way to the specimen surface the
light then passes through a Wollaston or Nomarski prism and is divided
into two orthogonal components, ordinary and extraordinary waves, with
the same amplitude. An optical shear occurs within the prism resulting in
a phase shift in each wavefront relative to the other. These phase shifts are
known as optical path differences or OPDs. Axial and lateral translation of
the Nomarski prism increases the shear. This increased shear increases the
three-dimensionality and coloration seen in the DIC image. The main dif-
ference between transmitted light and refl ected light DIC microscopy is that
only one prism is used with refl ected light while two are used in the trans-
mitted light system. After the light passes through the prism it is focused
onto the specimen surface where it interacts with the surface morphologies.
The light is then refl ected back up through the objective and passes through
an analyzer whose transmission direction is perpendicular to the polarizer.
In essence, the polarizer and analyzer are in the crossed polars orientation
during refl ected light DIC observations. Also the illuminating light should
be fairly collimated. This is accomplished by closing down the condenser
aperture diaphragm and the fi eld diaphragm of the Epi illuminator.
In refl ected light DIC microscopy, the optical path difference produced
by an opaque specimen is dependent upon the surface relief of the speci-
men and the phase retardation that results from the refl ection of sheared
and deformed orthogonal wavefronts by the surface. These optical path dif-
ferences are seen as differences in intensity and color in the eye piece. For
a majority of the specimens imaged with DIC, the surface relief varies only
within a relatively narrow height range. This height difference is usually on
the order of nanometers or micrometers. These specimen surfaces are usu-
ally observed as being fl at with very little variation in detail when observed
using an ordinary refl ected light stereo microscope.
Phase changes occurring at refl ection boundaries present in the speci-
men produce the optical path differences that lead to increased contrast
in the DIC image. These phase differentials are most likely to be found at
interfaces between different materials, such as grain boundaries and phase
transitions in metals and alloys, or aluminum and metal oxide regions in a
semiconductor integrated circuit. Many times highly convoluted polymer
surfaces such as non-woven fabrics, electrospun fi ber scaffolds and highly
