Phase Contrast Microscopy & Differential Interference Contrast Microscopy (DIC).pptx

1. Phase Contrast Microscopy: Introduction, Principle, Light path of Phase Contrast Microscope, Applications, Advantages, Limitations.

2. Differential Interference Contrast Microscopy (DIC):
Principle, Working, Application, Limitation.

1. Phase Contrast Microscopy

2. This microscopic technique is slightly different from the bright and dark field techniques because in this technique the light rays of different phases are combined and used to generate an image. Different components of cell (cell organelles) have different refractive indices (power to turn the light from its path). Due to this property, when the light passes through the cell, each cell part change the path of light according to its refractive indices, this create phase difference in light rays. This microscope is used for visualization of cell culture and live cells. Living cells can be observed without any staining.

3. Principle Phase contrast microscopy is based on the principle that small phase changes in the light rays, induced by differences in the thickness and refractive index of the different parts of an object, can be transformed into differences in brightness or light intensity

4. Light path of Phase contrast Microscope The light path consists of Light source Annular diaphragm Condenser Specimen stage Objective lens Phase plate Ocular lens

5. The light rays enter the annular diaphragm from its source This annular diaphragm blocks most of the light from the illuminator. Hence it produces a hollow cone of light. Hollow cone of light is focused on specimen before reaching the objective lens . Light travelling directly from the illuminator ( undiffracted wave) passes through one part of the phase plate whereas light refracted or scattered by the specimen (diffracted wave) passes through another part of the phase plate. This causes undiffracted wave to be out of phase with that of diffracted wave. As a result, diffracted and undiffracted rays formed different images.

6. Applications of Phase-contrast Microscopy To produce high-contrast images of transparent specimens, such as Living cells (usually in culture), Microorganims , This tissues slices, Subcellular particles (including nuclei and other organelles) Used in the study of dynamic cellular processes like bacterial spore formation and germination. It avoids the need to examine killed and fixed specimens.

7. Advantages The capacity to observe living cells. Specimens that not need to be killed, fixed or stained to view under a microscope. High contrast, high resolution images. Limitations Not ideal for thick organisms Limited to transparent or semi-transparent specimens .

8. Differential Interference Contrast Microscopy (DIC)

9. Principle Differential interference contrast microscopy (DIC) reveals cell organelles. DIC uses two beams of light to create high contrast, three dimensional images of the live specimens. DIC microscopy is similar to phase contrast in that it creates an image in detecting differences in refractive indices and thickness. It is a type of light microscopy that provides a detailed view of unstained live specimens by employing a polariser to produce polarized light.

10. Working Non Polarized from the light source is passed through a polarizer. Polarized light is produced. The polarized light then passes through a prism. Prism generates two distinct beams. These beams pass through the specimen and enter the objective lens. Here they are combined into one. An interference effect is produced, because the two beams pass through different substances with slightly different refractive indices and are not totally in phase. It gives three dimensional structures of the object.

11. Application and uses of DIC Microscopy The DIC microscopy is used to get three dimensional structures of the nucleus of eukaryotic cell, endospores, vacuoles, and granules. Interference microscope has a higher and better contrast. Limitations The equipment for DIC is quite expensive because of the many prisms that are required.