|
 Nomarski
or differential interference contrast (DIC) microscopy (invented by
Georges Nomarski in the mid-1950s) is used to image, living or stained
specimens, which contain little or no optical contrast when viewed using
brightfield illumination.
In transmitted light DIC,
light from the lamp is passed through a polariser located beneath the
condenser, in a manner similar to polarised light
microscopy. The polarised light then passes though a modified
Wollaston prism (below the condenser) that splits the entering beam of
light into two beams travelling in slightly different directions but
vibrating perpendicular to each other, and therefore unable to recombine
to cause interference. The distance between the two beams is called the
"shear" distance, and is always less than the resolving ability of the
objective, to prevent the appearance of double images. The split beams
enter and pass through the specimen where their paths are altered by the
specimen's varying thickness, slopes, and refractive indices. When the
parallel beams enter the objective, they are focused above the rear focal
plane where they enter a second modified Wollaston prism that recombines
the two beams at a defined distance outside of the prism. This removes the
shear and the original path difference between the beam pairs. However,
the parallel beams are no longer the same length because of path changes
caused by the specimen. In order for the parallel beams to interfere with
each other, the vibrations of the beams of different path length must be
brought into the same plane and axis. This is accomplished by placing a
second polariser (analyzer) above the upper Wollaston beam-combining
prism. The light then proceeds toward the eyepiece where it can be
observed as differences in intensity and colour. DIC microscopy causes one
side of an object to appear bright (or coloured) while the other side
appears darker (or a different colour). This shadow effect gives a pseudo
three-dimensional appearance to the specimen, but is not a true
representation of the geometry of the specimen, because it is based on
optical thickness. The pseudo 3D appearance of the specimen can also be
profoundly affected by it's orientation i.e. rotation of the specimen by
180 degrees can change a hill into a valley or vice versa. Therefore, DIC
microscopy is not suitable for accurate measurement of actual heights and
depths.
These are numerous advantages in DIC
microscopy as compared particularly to phase
microscopy. Using DIC microscopy it is possible to make fuller use of
the numerical aperture of the system because, unlike phase contrast
microscopy, there is no sub-stage annulus to restrict the aperture,
therefore, Köhler illumination is properly
utilized. Images can be seen in striking colour (optical contrast) with
a 3-dimensional shadowed-like appearance and at excellent resolution.
Use of the full objective aperture also enables the microscope to focus
on a thin plane section of a thick specimen without confusing images
from above or below the plane (optical sectioning). There are no
confusing halos, as may be encountered in phase images. DIC microscopy
is superb for investigating living cells, as it is non-invasive and
real-time, optical sectioning possibilities allow the movement of tiny
organelles to be followed with ease. There are however, several
disadvantages in DIC microscopy; the equipment for DIC is quite
expensive because of the many prisms that are required. Birefringent
specimens, such as those found in many kinds of crystals, may not be
suitable because of their effect upon polarised light. Similarly,
specimen carriers made of plastic, such as culture vessels, Petri dishes
etc, may not be suitable. For very thin or scattered specimens, better
images may be achieved using phase contrast methods.
|
