2. INTRODUCTION
• Georges (Jerzy) Nomarski (1919–1997) developed
modification of interference microscopes.
• Nomarski microscope is sometimes called a differential
interference contrast (DIC)microscope or a polarization
interference contrast microscope.
• The design of the Nomarski interference-contrast
microscope for transmitted light is described for two
different techniques.
• One for double-beam interference microscopy, and
compensation of interference fringes.
3. components of the basic differential
interference contrast microscope setup.
4. Interference of light
• Two waves superimpose to form a resultant waves of greater
or lower amplitude.
• Destructive Interference -Two or more than two waves -
sum of variations has smaller amplitude than component
variations.
• Constructive Interference- sum of variations will have
bigger amplitude than any of components individually
Constructive Interference Destructive Interference
5. Wollaston prism
• Invented by William Hyde Wollaston.
• Wollaston prisms - made of two layers of a crystalline
substance, such as quartz - due to the variation of
refractive index depending on the polarisation of the light,
splits the light according to its polarisation.
• It seperates randomly polaraised or unpolarised light into
two orthogonal linearly polarised outgoing beams.
6. NOMARSKI PRISM
It consists of two birefrigent crystal wedges.
• One of the wedges is identical to a conventional Wollaston wedge
and has the optical axis oriented parallel to the surface of the prism.
• The second wedge of the prism is modified by cutting the crystal in
such a manner that the optical axis is oriented obliquely with
respect to the flat surface of the prism.
• The Nomarski modification causes the light rays to come to a focal
point outside the body of the prism, and allows greater flexibility so
that when setting up the microscope the prism can be actively
focused.
7.
8. CONDENSER
• Main components of the optical system –condenser is a lens
concentrate light from illuminating source –focused through
the object &magnified by objective lens.
• The two rays are focused by the condenser for passage through the
sample. These two rays are focused so they will pass through two
adjacent points in the sample, around 0.2 μm apart.
• The sample is effectively illuminated by two coherent light sources,
one with 0 polarisation and the other with 90 polarisation.
9. CONTRAST
• The contrast is achieved by splitting the illuminating beam into
two beams displased by short distance on the sample surface
followed by reflection and reconstitution of the reflected beams.
Optical path length changes –change in the index of refraction
10. RESOLUTION OF OPTICAL
MICROSCOPE
• Resolution of an optical microscope is depends upon wavelenth of
illuminating light and the numerical aperture(NA) of the objective
lens.
R=0.61 /NA
• The resolution limit of the optical microscope is approimately0.25
µm.
11. Differential interference contrast (DIC)
Specimen
(inhomogen phase object)
Prism DIC prism
(Nomarski) (Nomarski)
Polarisator Analysator
Phase
difference
unpolarized linear two vertical linear polarized
light polarized polarized light
light waves (analysator vertical
vs. polarisator)
12. Differential interference contrast
microscopy
• DIC works by separating a polarised light
source into two orthogonally polarized
mutually coherent parts which are
spatially displaced (sheared) at the
sample plane, and recombined before
observation.
• The interference of the two parts at
recombination is sensitive to their optical
path difference (i.e. the product of
refractive index and geometric path
length).
• Adding an adjustable offset phase
determining the interference at zero
optical path difference in the sample, the
contrast is proportional to the path length
gradient along the shear direction.
13. • Light passes through a polariser and is reflected
downward toward birefringent crystals Wollaston prism .
Light is split into two mutually perpendicular polarsied
components that move at different velocities with an
angular divergence(d).
• After emerging from the prism and reflecting off the
sample -two beams recombine by passing once again
through the wollaston prism in the opposite direction.
• The reconstituted beams –passes through an analyser –
intensity changes observed .
.
14. • The polarised light enters the first Nomarski-modified
.
Wollaston prism and is separated into two rays polarised at
90 to each other, the sampling and reference rays.
• Microscope image contains contrast effects –depends on
difference in optical path length by changes in the
geometrical of the surface & difference in variation in index
of refraction-across the phase boundary
• Intensity variations seen-when monochromatic light
illuminates a substrate.
• Sample consists two phases with different refractive indices.
• Optical path differences between the two reflected light
beams –gives intensity variation .
• Interference contrast maximised in a direction parallel to the
maximum displacement of the two beams & zero in the
orthogonal direction
15. The micrographs are taken under identical polariser,analyser &
wollaston prism settings,but the sample(a) has been rotated
90˚ compared to (b).
• Interference contrast image in (a)
clearly shows ,when polariser,
analyser, & prism are adjusted to
maximum contrast.
• Contrast disappears –sample
rotates 90˚-extremely hard to
see(b).
• When the polarizer before the
prism, or the analyzer before the
detector, is rotated, the relative
intensities of the two orthogonal
polarized beams change, and the
colors and contrast change.
16. Advantages and disadvantages
• Better resolution-than other optical microscope
• The main limitation of DIC is its requirement for a
transparent sample of fairly similar refractive index to its
surroundings. DIC is unsuitable (in biology) for thick
samples, such as tissue slices, and highly pigmented
cells.
• DIC is also unsuitable for most non biological uses
because of its dependence on polarisation, which many
physical samples would affect.
