Differential

Interference
Contrast Microscope
 Problems With Phase Contrast
Microscope
◈ Despite various advantages, following are two main disadvantages
of Phase Contrast Microscope:
- Halo effect
- Shade off effect
 What is Halo Effect?
◈ It is described as appearance of bright or dark edges around large
specimens.
◈ This occurs because some of the diffracted light from specimen
can cross the phase ring, which leads to incomplete separation of
diffracted light from background light.
◈ Halos are bright for positive phase contrast and dark for negative
phase contrast.
 Shade off Effect
◈ It is a situation where homogenous parts of specimen are
displayed with same light intensity.
◈ Although the light passing through the regions experiences a phase
shift, only minor diffraction occurs.
◈ Therefore, the light waves enter phase ring like undeviated and do
not experience interference.
Normal path of diffracted and
undiffracted beams in Phase
contrast Microscope
 Introduction and Principle of DIC Microscope
◈ Georges Nomarski developed this method in 1960.
◈ Differential Interference Contrast (DIC) is a microscopy technique that
introduces contrast to images of specimens which have little or no
contrast when viewed using brightfield microscopy.
◈ It is based on an older method of interferometry (waves are superimposed
which cause phenomenon of interference).
◈ It is similar to phase contrast microscope in that it creates image by
detecting differences in refractive indices.
◈ A beam of light is split into two, one of which passes through specimen
(object beam) and the other passes through clear area of slide
(reference beam). The beams are then recombined to produce an
interference pattern. The recombination of two beams produced an
enhanced image of specimen.
 Parts of DIC Microscope
◈ Light source- It generates polychromatic light.
◈ Polarizer- Inserted between the incandescent source of illumination and
condenser, it is designed to produce plane polarised light.
◈ Nomarski Prism- Also called Wollaston prism or condenser prism or beam-
splitting prism, it splits the plane polarized beam into two beams. One
beam passes through the specimen and is called specimen beam. The other
beam passes through slide/background and is called reference beam.
◈ Objective Prism- It recombines the separated beams.
◈ Analyzer- Interference occurs here which produces differential
interference contrast.
◈ Eye Piece- Image is observed.
Parts of DIC Microscope
 Working of DIC Microscope
◈ The visible light from light source passes through polarizer and is
gets converted to polarized light.
◈ The beam of plane polarized light, passes through Nomarski prism
and is separated into two beams.
◈ One beam is directed through the specimen and is called object
beam. The other passes through background/slide and is called
reference beam.
◈ The two beams are combined by an upper Nomarski prism.
◈ The combined light passes through the analyser and then eye piece.
 Advantages and applications
◈ Its clarity is better than standard optical microscope.
◈ It can be used to examine live and unstained specimens.
◈ Where phase contrast microscope reveals only sharp
discontinuities, DIC permits detection of small and continuous
changes.
◈ Halos around structures are not seen.
◈ It is used widely in-
- Imaging fibrous structure of nerve or muscles
- Imaging mitotic spindles
- Observation of crystal growth process
Comparison between image formed by DIC
and Phase Contrast Microscope

Illustrated in Figure 1 are several digital images comparing specimens captured in DIC and phase contrast. A human buccal mucosa
epithelial (cheek) cell, revealing the nucleus, cytoplasmic inclusions, and numerous bacteria on the upper surface, is presented in Figure
1(a), imaged with differential interference contrast. The same viewfield with phase contrast illumination is illustrated in Figure 1(b). In
Figure 1(c), a thick section of murine kidney tissue imaged with differential interference contrast reveals a bundle of cells enclosed within
a tubule. A phase contrast image (Figure 1(d)) of the same area is confusing and disturbed by the presence of phase halos outside the plane
of focus. Relatively high magnification views of an Obelia polypoid annulated stem perisarc in DIC and phase contrast are illustrated in
Figures 1(e) and 1(f), respectively. In DIC, (Figure 1(e)) the annular structure appears hemispherical with internal rays that emanate
radially from the stem. In addition, granular particles are visible within the stem structure, but anatomical detail is largely undefined. The
phase contrast image (Figure 1(f)) is confused by halos around the annular rings and within the stem.
Fig 4(a) & (b) illustrate human erythrocytes as observed under PCM and DIC microscope
respectively.
Fig 4(c) & (d) illustrate adherent HeLa cells as observed under PCM and DIC Microscope
respectively.
Fig 4(e) & (f) illustrate Zygnema algae as observed under PCM and DIC microscope