NOMARSKI INTERFERENCE CONTRAST OPTICAL MICROSCOPY

M.SARAVANAKUMARNANOSCIENCE AND TECHNOLOY

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.

components of the basic differential interference contrast microscope setup.

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

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.

NOMARSKI PRISMIt 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.

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.

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

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.

Differential interference contrast (DIC)

Specimen(inhomogen phase object)

Phasedifference

Polarisator

linearpolarized

light

Prism(Nomarski)

two verticalpolarized

waves

Analysator

DIC prism(Nomarski)

linear polarizedlight

(analysator verticalvs. polarisator)

unpolarizedlight

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.

• 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 .

.

.• 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

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.

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.

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