Dark Field microscopy

it will be helpful in understanding dark field microscopy and its advantages and disadvantages.

1. Topic : Dark field microscopy Presented by : Rafia Altaf

2. Contents What is dark field microscopy? Principle and working Applications Advantages & disadvantages

3. What is dark field microscopy? Definition: Dark field microscopy produces an image with a dark background . Dark field microscopy is a technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background. used to examine live microorganisms that are not visible with light microscope and cannot stained, or distorted by staining and cannot be identified .

4. Conti…. Instead of normal condenser, dark field microscope uses dark field condenser that contain a opaque disc. This disk blocks light that would enter the lens directly, only the light is reflected off the specimen enter objective lens. As there is no background light, the specimen appears light the against black background the dark field.

5. Schematic diagram:

6. Principle: it creates a contrast between the object and the surrounding field e.g the background is dark and the object is bright. the objective and the ocular lenses used in the dark ground microscope are same as that of ordinary light microscope. 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. Oblique illumination was the first step toward dark field microscopy. In dark field, light seems to radiate from the specimen while all the rest of the field is black. Resolution is as good as that in bright field while contrast is enhanced.

7. Conti…..

8. Dark field condensers: High Numerical Aperture Dark Field Condensers : The first condenser made specifically for dark field was produced by Francis H. Wenham and George Shadbolt in 1855 . This condenser used a parabolic glass reflector to create a hollow cone of light. Unlike a refracting condenser, a reflecting condenser does not produce chromatic aberrations and a parabolic shape minimizes spherical aberrations. The result is a more finely focused spot of light.

9. High Numerical Aperture Dark Field Condensers :

10. Low Magnification Dark Field Condensers A low magnification dark field condenser can be nothing more than an ordinary bright field condenser with an opaque disk of the proper diameter placed in its front focal plane. The diameter of the opaque disk must be just large enough to prevent any direct light from entering the objective.

11. Types of Specimens for Dark Field Very thin histological sections can be used if unstained or if only certain components are stained, as in silver stains. Biological fluids from animals and plants, cell cultures, microbes, foods, fibers, crystals, colloids, and sub-microscopic particles are all suitable for dark field microscopy.

12. Uses: Viewing blood cell viewing bacteria Viewing different type of algae Viewing hair line metal fracture Viewing diamonds and other precious stones

13. DARK-FIELD SMARTPHONE MICROSCOPE WITH NANOSCALE RESOLUTION FOR MOLECULAR DIAGNOSTICS Recently, fluorescence-based nanoparticle detection was reported using Smartphone adding nanotechnology (NT) to Smartphone-based imaging methods

14. Smartphone:

15. Advantages: It is ideal for viewing unstained objects, transparent and absorb little or no light. These specimens often have similar refractive indices as their surrounding, making them hard to distinguish with other illumination techniques. Used in research of live bacterium, as well as mounted cells and tissues. Useful in examining external details, edges and surface defects. Dark field has regained its popularity when combined with other illumination techniques, such as fluorescence, which widens its possible employment in certain field.

16. Disadvantage: not good for internal structure -Dust on sample, optics, bubbles in oil are not tolerated with this technique If oil or water on the condenser or slide, air bubble will produce.

17. REFERENCES Q. Wei, H. Qi , W. Luo , D. Tseng, S. J. Ki , Z. Wan, Z. Göröcs , L. A. Bentolila , T. Wu, R. Sun, and A. Ozcan , “Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone,” ACS Nano , 7, (10), pp 9147-9155, 2013. http://www.microscormaster.com.dark-field-miroscopy,html http://public.wse.edu/omoto/papers/darkfield.html http://www,microscopeworld.com/t-darkfield-microscopy

18. Thank you……!