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What is dark field illumination?

What is Darkfield Microscopy?

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Darkfield microscopy is a special form of microscopy in which the light beam is split in such a way that the edges of objects in the samples are illuminated so that they appear as silhouettes against a dark background — as opposed to brightfield microscopy which allows the examination of specimens against an illuminated field — and which washes out the tiny and faint objects that can be seen only in darkfield. The second major difference between darkfield and other forms of microscopy is that darkfield can be used to view wet samples, including live blood and other liquids or apparently liquid substances.

Because of the differences in illumination, there are many features in samples that are only viewable in darkfield and never seen in other kinds of microscopy. It is probably for this reason that some of the findings of darkfield microscopists are rejected by those who also examine slides but never see the objects reported by darkfield specialists.

Darkfield microscopy is not new. However, to put everything in context, it might be worth noting that magnification of objects has fascinated and challenged many careful observers for countless centuries. Anton van Leeuwenhoek (1632-1723) is generally credited with the invention of the microscope, but it took his successors 150 years to match the quality Leeuwenhoek had managed with much simpler optics.

Likewise, Royal Raymond Rife’s microscopes of more than half a century ago remain unrivaled today, this despite the advent of fiber optic illumination and other advances that, all other things equal, should have furthered the development of improved microscopes.

Points to Understand

The splitting of the light beam is achieved by blocking the light from coming up straight through the condenser. This little obstacle causes the light to refract and appear to come from the edges. Because darkfield permits the observer to see liquid samples, no stains are required and the objects in the sample may live for many days following removal from their source. So, in addition to being able to see objects that are not visible in brightfield, darkfield microscopy facilitates the study of behavioral patterns that cannot be observed with stained or fixed specimens.

Since what we understand is often as not based on what we see, it goes without saying that opinions about blood, immunity, germs, and illness can be permanently transformed after only a few hours of darkfield viewing.

The ramifications of this statement are so vast that it will probably be wise to allow the understanding and appreciation of darkfield microscopy a little time to unfold and mature. However, before doing so, let me simply make a couple of comments:

The idea that blood is sterile is based on the inability to see what is floating between the “recognized” blood components such as red blood cells, white blood cells, and platelets.
An entire century of medicine was based on theories of germs and germ transmission that are tied to observations that are limited and possibly dubious.

darkfield blood analysis

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What is Advantages of dark field illumination?

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 illumination 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.

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What is Advantages of dark field illumination?

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 illumination 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 illumination?

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 dark field illumination Applications?

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

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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