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DARK FIELD MICROSCOPY

PRESENATION BY: GIDWANI MANISH N.1522964

SOURCE: http://www.photomacrography.net/forum/userpix/1469_DSC_4395_2.jpg

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CONTENTS.

• WHAT IS DARK FIELD MICROSCOPY?• HOW STUFF WORKS…?• TRANSFORMATION.• TOO EXPENSIVE? HERE’S WHAT YOU CAN DO..• APPLICATIONS, ADVANTAGES &

DISADVANTAGES.• EVIDENCE FOR USE IN RESEARCH IMAGING.• REFERENCES.

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WHAT IS DARK FIELD MICROSCOPY?

Dark Field Microscopy is a technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.

PIC: Highly magnified image of sugar crystals using darkfield microscopy technique.

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HOW STUFF WORKS…?

SOURCE: http://www.gonda.ucla.edu/bri_core/mic3.gif

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LIGHT DIAGRAMS.

CHARLIESOURCE: http://secure.tutorsglobe.com/CMSImages/1006_phase%20contrast%20microscope.jpg

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TRANSFORMATION.

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TOO EXPENSIVE? HERE’S WHAT YOU CAN DO..

• If you do not have access to “STOP” accessories and cannot afford a dark field kit, there are alternative ways to adapt your microscope for dark field illumination.

• The expensive stops are all made of opaque material.• One option is to use a circular object, such as a coin; adhere the

coin to a larger disk and place below the stage.• You can also cut out a round piece of thick paper, such as

construction paper, cardboard or poster-board, and attach to the condenser.

• Whatever you use, the trick is to find the right diameter so that the makeshift stop will block the light and only allow the oblique rays to illuminate the specimen.

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APPLICATIONS.• Viewing blood cells (biological dark field microscope, combined

with phase contrast)• Viewing bacteria (biological dark field microscope, often

combined with phase contrast)• Viewing different types of algae (biological dark field microscope)• Viewing hairline metal fractures (metallurgical dark field

microscope)• Viewing diamonds and other precious stones (gemological

microscope or stereo dark field microscope)• Viewing shrimp or other invertebrates (stereo dark field

microscope)

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ADVANTAGES & DISADVANTAGES.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.

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 artifacts 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.

• Dark field needs an intense amount of light to work. This, coupled with the fact that it relies exclusively on scattered light rays, can cause glare and distortion.

• It is not a reliable tool to obtain accurate measurements of specimens.

• Finally, numerous problems can arise when adapting and using a dark field microscope. The amount and intensity of light, the position, size and placement of the condenser and stop need to be correct to avoid any aberrations.

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EVIDENCE FOR USE IN RESEARCH IMAGING.

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SOME MORE EVIDENCE…

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REFERENCES.• http://www.microscopemaster.com/dark-field-microscope.html• http://public.wsu.edu/~omoto/papers/darkfield.html• https://www.microscopeworld.com/t-darkfield_microscopy.aspx• http://www.gonda.ucla.edu/bri_core/mic3.gif• http://secure.tutorsglobe.com/CMSImages/1006_phase%20contrast

%20microscope.jpg• http://www.photomacrography.net/forum/userpix/1469_DSC_4395_2.jpg• Robert M. Macnab (3/5/1976): “Examination of Bacterial Flagellation by

Dark-Field Microscopy.” Journal Of Clinical Microbiology, Sept. 1976, p. 258-265. vol. 4, No. 3.

• Sergiy Patskovsky, Eric Bergeron, David Rioux, and Michel Meunier (26/6/2014): “Wide-field hyperspectral 3D imaging of functionalized Gold nanoparticles targeting cancer cells by reflected light microscopy.” J. Biophotonics 1–7 (2014)/DOI 10.1002/jbio.201400025.

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THANK YOU.