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advantages and disadvantages of dark field microscopy

Disadvantages Dark Field Microscopy

Darkfield microscopy is very sensitive to dirt and dust located in the light path.
It is not suitable for all specimens. If the refractive index of a transparent specimen is similar to the surrounding medium, then the specimen light will pass right through the specimen and it will not be scattered into the objective.
The intensity of the illumination system must be high so see the specimen properly.
It is necessary to open the condenser aperture diaphragm, and this limits the effective use of the diaphragm.
One patch stop is generally sufficient for low magnification work, but at a higher magnification the quality of the image drops. It may be necessary to experiment with different patch stop sizes for the different objectives.

The main limitation of dark-field microscopy is the low light levels seen in the final image. This means that the sample must be very strongly illuminated, which can cause damage to the sample. Dark-field microscopy techniques are almost entirely free of artifacts, due to the nature of the process. However, the interpretation of dark-field images must be done with great care, as common dark features of bright-field microscopy images may be invisible, and vice versa.

While the dark-field image may first appear to be a negative of the bright-field image, different effects are visible in each. In bright-field microscopy, features are visible where either a shadow is cast on the surface by the incident light or a part of the surface is less reflective, possibly by the presence of pits or scratches. Raised features that are too smooth to cast shadows will not appear in bright-field images, but the light that reflects off the sides of the feature will be visible in the dark-field images.

Advantage Dark Field Microscopy


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.

Dark-field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-borne, single-celled organisms. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.

It is a simple procedure which can be used on live transparent specimens, specimens which normally need to be stained (and therefore killed).

The images appear spectacular and are visually impressive.

Darkfield microscopy even allows for the visualization of objects that are below (!) the resolution of the microscope. These objects will appear as bright spots on a dark background. It is not possible to see the shape of these objects, however.

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.

Dark Field Microscopy Blood Images





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