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What is  dark field microscopy ?

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What is a Dark Field Microscopy? The dark field microscopic examination of freshly collected, vital blood is a pillar of the Paracelsus Clinica al Ronc holistic medical diagnosis. It provides information on the internal milieu and function of the blood cells, as well as the amount and development of endobionts, from which microorganisms and more sophisticated structures, such as bacteria, fungi, and viruses, develop.

What is Dark Field Microscopy?-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.

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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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advantages of dark field microscopy advantages of dark field microscopy

What advantages of dark field microscopy?

Dark Field Microscopy Microscopy disadvantages are-While Dark Field Microscopy can create beautiful images under the right circumstances, there are a number of disadvantages to Dark Field Microscopy Microscopy:

1. Dark Field Microscopy needs an intense amount of light to work. This intense light can create glare and distortion. Therefore, Dark Field Microscopy does not create reliable specimen measurements.

2. Dark Field Microscopy is sensitive to contaminants. You need to be meticulous about cleaning your specimen slides and all optical surfaces when performing Dark Field Microscopy imaging, as every speck of dirt will want to light up when using Dark Field Microscopy. You also need to be careful when preparing your specimens, as any contaminates (dust, air bubbles, etc) in your mounting, above or below the plane of focus, can degrade your image.

3. Specimens which are not thin enough are prone to degradation and distortion. The best Dark Field Microscopy specimens should be thin to reduce diffraction artifacts.

Because of Dark Field Microscopy Microscopy’s limitations and recent advances in microscopy techniques (such as phase contrast and DIC) Dark Field Microscopy is not used often in modern imaging. However, Dark Field Microscopy is seeing a resurgence in popularity as it is combined with other techniques such as fluorescence microscopy.

Limited colors (certain colors will appear, but they’re less accurate and most images will be just black and white)

Images can be difficult to interpret to those unfamiliar with Dark Field Microscopy Microscopy

Although surface details can be very apparent, the internal details of a specimen often don’t stand out as much with a Dark Field Microscopy setup.

Dark Field Microscopy Microscopy advantages-No one system is perfect, and Dark Field Microscopy Microscopy may or may not appeal to you depending on your needs.

Some advantages of using a Dark Field Microscopy microscope are:

Extremely simple to use

Inexpensive to set up (instructions on how to make your own Dark Field Microscopy microscope are below)

Very effective in showing the details of live and unstained samples

What Application of dark field microscopy?

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