Differential Interference Contrast Microscope.pptx

Differential Interference Contrast Microscope

1. Nomarski Differential Interference Contrast Microscope Submitted by- Aditya Sharma Class- Msc . Biotechnology Semester-1 Submitted to- Dr. Ashima Pathak Roll no. 2084009

2. Problems With Phase Contrast Microscope Despite various advantages, following are two main disadvantages of Phase Contrast Microscope: Halo effect Shade off effect

3. 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.

4. 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.

5. Normal path of diffracted and undiffracted beams in Phase contrast Microscope

6. 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.

7. 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.

8. Parts of DIC Microscope

9. 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.

10. 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

11. 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.

12. 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 respectively.

13. Differences between Phase Contrast Microscope and DIC Microscope Phase Contrast Microscope Differential Interference Contrast Microscope Developed by Zernike. Developed by Nomarski . Works by detection of sharp changes in refractive index. Works by detection of continuous change of refractive index. Utilizes two beams without beam splitters. Utilizes two beams with beam splitters. Polarizers are not used. Polarizers are used. Uses annular diaphragm and phase plates to create contrast Annular diaphragm and phase plate are not used. Halo around images are produced. Halos are not observed. Cost is moderate. Cost is high. No 3-Dimensional effect is seen. Images are produced with pseudo 3-D effect.

14. Path of Light in Phase Contrast Microscope Path of light in DIC Microscope

15. References Bagnell Robert, Differential Interference Contrast Microscopy, In: Pathology-464-Light microscopy; 2012. p. 1-9 Willey, Sherwood, Woolverton ; The Study of Microbial Structure: Microscopy and Specimen Preparation, In: Prescott, Harley and Klein’s Microbiology ; 2008. p. 23 Pathak Ashima; Phase Contrast Microscope, In: Fundamentals of Microscope; . p. 64-68 Sarvankumar M. (2012) ‘ Nomarski Interference Contrast Optical Microscopy’ [PowerPoint presentation] Available at: https://www.slideshare.net/SARAVANA3061987/nomarski-dic (Accessed: 20 January 2021) Eberhard Christian (2012). Development of a Model System to Study Cell Adhesion and Cell Mechanics

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