Interference microscopy

Interference Microscopy
Over the past few years the microscopic techniques had faced with some complications with the observation of living specimens as transparency, light and because of that don’t have sufficient contrast. Thus with the development of these techniques was possible to visualize specimens with adequate contrast.

The Interference Microscopy or Quantitative Interference Microscopy is one of these techniques that derive from Phase Contrast Microscopy but is more sensitive than this technique and make possible the easy and clarify viewing of living organisms.

This technique is used by taking light from a condenser and using a prism to split the light into two beams. One beam, object beam, passes through the specimen and the objective. The other beam, reference beam, passes through another objective without touching the specimen. These beams allow a specimen to be seen through the difference in the fields caused by the two beams and the differences of the two images allow details to be seen.

There is a variation of interference microscopy called Differential Interference Contrast microscopy (DIC), also known as Nomarski Interference Contrast microscopy (NIC) or simply Nomarski microscopy. This optical microscopy illumination technique used to enhance the contrast in unstained or transparent samples was named after its inventor and also uses two beams produced by a single polarized light.

Initially the polarised light enters in the first Nomarski-modified Wollaston prism and is separated into two rays polarised to each other, the sampling and reference rays. Then this two rays are focused by the condenser for passage through the sample and travel through adjacent areas of the sample, separated by the shear (separation is normally similar to the resolution of the microscope).

After that they will experience different optical path lengths where the areas differ in refractive index or thickness which causes a change in phase of one ray relative to the other due to the delay experienced by the wave in the more optically dense material.

Lastly the rays travel through the objective lens and are focused for the second Nomarski-modified Wollaston prism which recombines the two rays into one polarized which make a image with a three-dimensional appearance. This final combination of rays leads to interference, brightening or darkening the image at that point according to the optical path difference.

These interference techniques have strong advantages in uses involving living or unstained biological samples, specially their applications in biology, crystallography, mineralogy and chemistry. And also, its resolution and clarity in conditions such as this are unrivaled among standard optical microscopy techniques.

On the other hand the main limitation of these techniques is its requirement for a transparent sample of fairly similar refractive index to its surroundings. Differential Contrast Microscopy is also unsuitable for thick samples (like tissue slices and highly pigmented cells) and for most non biological uses because of its dependence on polarization, which many physical samples would affect.