Dark field microscopy

Dark field Microscopy working advantages and disadvantages

1. Dark Field Microscopy By:- Abhishek Rajesh Indurkar 17PBT202

2. Contents What is dark field microscopy? Principle and working Applications Advantages Disadvantages References

3. What is dark field microscopy? A dark field microscopy is used to examine live micro-organisms that either invisible in the ordinary light microscope, cannot be stained by standard methods, or are so distorted by staining that their characteristics then cannot be identified. Instead of the normal condenser, a dark field microscope uses a dark field condenser that contain a opaque disc. The disc blocks light that would enter the lens directly, only the light is reflected off the specimen enters the objective lens. Because there is no background light, the specimen appears light against black background- the dark field.

4. Principle The dark ground microscope creates a contrast between the object and the surrounding field, such that, the background is dark and the object is bright. The objective and the ocular lenses used in the dark ground microscope are the same as in the ordinary light microscope, however, a special condenser is used, which prevents the transmitted light from directly illuminating the specimen. Only oblique scattered light reaches the specimen and passes onto the lens system causing the object to appear bright against a dark background.

5. Working

6. Applications • Used for diagnostic of Syphilis ( Treponema pallidum ). • Viewing blood cells. • Viewing bacteria. • Viewing different types of algae. • Viewing hairline metal fracture. • Viewing diamonds and other precious stones. • Viewing shrimp or other invertebrates.

7. Advantages • A dark field microscope is ideal for viewing objects that are unstained, transparent and absorb little or no light. • These specimens often have similar refractive indices as their surroundings, making them hard to distinguish with other illumination techniques. • You can use dark field to study marine organisms such as algae and plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some ceramics. • You can also use dark field in the research of live bacterium, as well as mounted cells and tissues. • It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure. • Dark field microscopy is often dismissed for more modern observation techniques such as phase contrast and DIC, which provide more accurate, higher contrasted images and can be used to observe a greater number of specimens. • Recently, dark field has regained some of its popularity when combined with other illumination techniques, such as fluorescence, which widens its possible employment in certain fields.

8. Disadvantages First, dark field images are prone to degradation, distortion and inaccuracies. • A specimen that is not thin enough or its density differs across the slide, may appear to have artefacts throughout the image. • The preparation and quality of the slides can grossly affect the contrast and accuracy of a dark field image. • You need to take special care that the slide, stage, nose and light source are free from small particles such as dust, as these will appear as part of the image. • Similarly, if you need to use oil or water on the condenser and/or slide, it is almost impossible to avoid all air bubbles. • These liquid bubbles will cause images degradation, flare and distortion and even decrease the contrast and details of the specimen. • It is not a reliable tool to obtain accurate measurements of specimens.

9. Reference http://medind.nic.in/ibo/t08/i2/ibot08i2p105.pdf http://www.battlesnake.co.uk/_uni/articles/holo/4-darkfield.pdf http://nptel.ac.in/courses/102103044/pdf/mod3.pdf https://toutestquantique.fr/en/dark-field-and-phase-contrast/ https://www.cdc.gov/std/syphilis/manual-1998/chapt5.pdf

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