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Dark field microscopy
- 1. DARK FIELD MICROSCOPY PRESENATION BY: GIDWANI MANISH N. 1522964 CHARLIE 1 SOURCE: http://www.photomacrography.net/forum/userpix/1469_DSC_4395_2.jpg
- 2. 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. CHARLIE 2
- 3. 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. CHARLIE 3 PIC: Highly magnified image of sugar crystals using darkfield microscopy technique.
- 4. HOW STUFF WORKS…? CHARLIE 4 SOURCE: http://www.gonda.ucla.edu/bri_core/mic3.gif
- 7. 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. CHARLIE 7
- 8. 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) CHARLIE 8
- 9. 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 condenserCHARLIE 9
- 10. CHARLIE 10
- 11. EVIDENCE FOR USE IN RESEARCH IMAGING. CHARLIE 11
- 12. SOME MORE EVIDENCE… CHARLIE 12
- 13. 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%20 microscope.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. CHARLIE 13
- 14. CHARLIE 14
Editor's Notes
- When light hits an object, rays are scattered in all azimuths or directions. The design of the dark field microscope is such that it removes the dispersed light, or zeroth order, so that only the scattered beams hit the sample.The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object. The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.
- Darkfield microscopy relies on a different illumination system. Rather than illuminating the sample with a filled cone of light, the condenser is designed to form a hollow cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again into a hollow cone. The objective lens sits in the dark hollow of this cone; although the light travels around and past the objective lens, no rays enter it The entire field appears dark when there is no sample on the microscope stage; thus the name darkfield microscopy. When a sample is on the stage, the light at the apex of the cone strikes it. The image is made only by those rays scattered by the sample and captured in the objective lens (note the rays scattered by the specimen in Figure 1). The image appears bright against the dark background. This situation can be compared to the glittery appearance of dust particles in a dark room illuminated by strong shafts of light coming in through a side window. The dust particles are very small, but are easily seen when they scatter the light rays. This is the working principle of darkfield microscopy and explains how the image of low contrast material is created: an object will be seen against a dark background if it scatters light which is captured with the proper device such as an objective lens.
- 1.Light enters the microscope for illumination of the sample. 2.A specially sized disc, the patch stop (see figure) blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can also be reasonably substituted at low magnification. 3.The condenser lens focuses the light towards the sample. 4.The light enters the sample. Most is directly transmitted, while some is scattered from the sample. 5.The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct illumination block (see figure). 6.Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.
- If a microscope has built-in elements to easily modify for dark field illumination, the manufacturer usually lists this amongst the observation specifications.You can achieve dark field by using condensers, mirrors and/or a “stop.” Some microscopes come with these accessories or researchers can purchase dark field kits, or even use some common items to adapt a microscope for dark field illumination.In bright field illumination, the object is lit from below the stage, resulting in a larger, contrasted image that can be studied.A dark field microscope blocks this central light with a condenser so that only oblique rays hit the object.An Abbe condenser, for example, contains a concave orb that collects light rays in all azimuths that bounce off a sample to form a cone of illumination.If there is nothing on the stage, the aperture of the condenser is greater than the objective and the view will be completely black.A stop is an opaque object that blocks the central light when placed underneath the stage condenser.This also causes light to scatter in all azimuths, resulting in a cone of light that allows for dark field observation.
- The above image, taken by Dr. Arlene Wechezak, won second place. The photographed specimen is the red algae Scagelia, showing reproductive spores and golden diatoms. The technique used to take the shot, dark field microscopy, removes the background by illuminating the sample with light that won’t be collected by the objective lens, creating a very dark background with the sample illuminated in the foreground.
- LEFT:(A) S. typhimurium during translational movement. Theperitrichous flagella form apolar bundle (see cartoon, Fig. 1B) which could be mistaken forpolarflagellation. Classifications should therefore be based on the appearance offlagella on stationary cells. Dark-fieldphotomicrograph taken with high-intensity pulsed xenon arc. Bar equals 5 gm. RIGHT: Polar flagellation in pseudomonads. (A) Stationary cell ofP. stutzeri. (B) Same negative [overex- posed duringprinting] to show cell outline. (C) Cartoon combining information from (A) and (B). (D-F) As for (A-C) but with P. aeruginosa (note presence oftwo flagella at pole, although species is predominantly monotrichous). Bar equals 5 ,um.
- LEFT: Microscopy images of fixedCD44+-MDA-MB-231 cells incubatedwith CD44-targetedAuNPs taken at differ-ent planes along the optical z-axis (1 μm distance step) in darkfield illumination mode (A1–4) and in reflected light imagingmode (B1–4). For better clarity an embossing filter is applied for imagesA2,A4, B2, B4. RIGHT: