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Wha is dark field microscopy?

What is dark field microscopy?

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All of us are quite familiar with the appearance and visibility of stars on a dark night, this despite their enormous distances from the Earth. Stars can be readily observed at night primarily because of the stark contrast between their faint light and the black sky.

Yet stars are shining both night and day, but they are invisible during the day because the overwhelming brightness of the sun “blots out” the faint light from the stars, rendering them invisible. During a total solar eclipse, the moon moves between the Earth and the sun blocking out the light of the sun and the stars can now be seen even though it is daytime. In short, the visibility of the faint star light is enormously enhanced against a dark background.

This principle is applied in darkfield (also called darkground) microscopy, a simple and popular method for making unstained transparent specimens clearly visible. Such objects often have refractive indices very close in value to that of their surroundings and are difficult to image in conventional brightfield microscopy. For instance, many small aquatic organisms have a refractive index ranging from 1.2 to 1.4, resulting in a negligible optical difference from the surrounding aqueous medium. These are ideal candidates for dark field microscopy.

dark field microscopy requires blocking out of the central light which ordinarily passes through and around (surrounding) the specimen, allowing only oblique rays from every azimuth to “strike” the specimen mounted on the microscope slide. The top lens of a simple Abbe darkfield condenser is spherically concave, allowing light rays emerging from the surface in all azimuths to form an inverted hollow cone of light with an apex centered in the specimen plane. If no specimen is present and the numerical aperture of the condenser is greater than that of the objective, the oblique rays cross and all such rays will miss entering the objective because of their obliquity. The field of view will appear dark.

The darkfield condenser/objective pair illustrated in Figure 1 is a high-numerical aperture arrangement that represents darkfield microscopy in its most sophisticated configuration, which will be discussed in detail below. The objective contains an internal iris diaphragm that serves to reduce the numerical aperture of the objective to a value below that of the inverted hollow light cone emitted by the condenser. The cardioid condenser is a reflecting darkfield design that relies on internal mirrors to project an aberration-free cone of light onto the specimen plane.

When a specimen is placed on the slide, especially an unstained, non-light absorbing specimen, the oblique rays cross the specimen and are diffracted, reflected, and/or refracted by optical discontinuities (such as the cell membrane, nucleus, and internal organelles) allowing these faint rays to enter the objective. The specimen can then be seen bright on an otherwise black background. In terms of Fourier optics, dark field microscopy removes the zeroth order (unscattered light) from the diffraction pattern formed at the rear focal plane of the objective. This results in an image formed exclusively from higher order diffraction intensities scattered by the specimen.

The photomicrographs in Figure 2 illustrate the effects of darkfield and brightfield illumination on silica skeletons from a small marine protozoan (radiolarian) in a whole mount specimen. In ordinary brightfield, skeletal features of the radiolarian are not well defined and tend to be washed out in photomicrographs recorded either with traditional film or digitally captured. was taken in brightfield illumination with the condenser aperture diaphragm closed to a point where diffraction artifacts obscure some of the sample detail. This enhances specimen contrast at the expense of image distortion. Under dark field microscopy, more detail is present, especially in the upper portion of the organism, and the image acquires an apparent three-dimensional appearance. When a red filter is used in conjunction with a darkfield stop , the radiolarian takes on a colorful appearance that is more pleasing, although no additional detail is produced and there is even some reduction in image quality.

Specimens that have smooth reflective surfaces produce images due, in part, to reflection of light into the objective. In situations where the refractive index is different from the surrounding medium or where refractive index gradients occur (as in the edge of a membrane), light is refracted by the specimen. Both instances of reflection and refraction produce relatively small angular changes in the direction of light, allowing some to enter the objective. In contrast, some light striking the specimen is also diffracted, producing a 180-degree arc of light that passes through the entire numerical aperture range of the objective.

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What Advantages of dark field microscopy?

 

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.

What Disadvantages of dark field microscopy

A dark field microscope can result in beautiful and amazing images; this technique also comes with a number of 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.
Dark field has many applications and is a wonderful observation tool, especially when used in conjunction with other techniques.

However, when employing this technique as part of a research study, you need to take into consideration the limitations and knowledge of possible unwanted artifacts.

What is Conclusion about dark field microscopy?

A dark field microscopy can offer brilliant, light images against a dark background of otherwise difficult to view specimens.Most standard microscopes come with dark field capabilities or accessories to enable this illumination technique.There are many practical applications of dark field, especially in the field of marine biology, in viewing the many specimens you cannot see using alternative techniques.However, a researcher must keep in mind the potential issues and limitations that may arise from dark field illumination.For further information, check out the many microscopy imaging techniques available.

What dark field microscopy Applications?

Viewing blood cells (biological dark field microscopy, combined with phase contrast)
Viewing bacteria (biological dark field microscopy, often combined with phase contrast)
Viewing different types of algae (biological dark field microscopy)
Viewing hairline metal fractures (metallurgical dark field microscopy)
Viewing diamonds and other precious stones (gemological microscope or stereo dark field microscopy)
Viewing shrimp or other invertebrates (stereo dark field microscopy)

In darkfield microscopy, contrast is created by a bright specimen on a dark background. It is ideal for revealing outlines, edges, boundaries, and refractive index gradients but does not provide a great deal of information about internal structure. Ideal subjects include living, unstained cells (where darkfield illumination provides information not visible with other techniques), although fixed stains cells can also be imaged successfully. Darkfield imaging is particularly useful in haematology for the examination of fresh blood. Non-biological specimens include minerals, chemical crystals, colloidal particles, inclusions and porosity in glass, ceramics, and polymer thin sections.

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