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What is dark field microscopy?

working principle of dark field microscopy

What is Dark field Microscopy?-Dark field 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.

Dark field Microscopy 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.

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Dark field 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.

Dark field Microscopy 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.

The Dark field Microscopy 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, Dark field Microscopy facilitates the study of behavioral patterns that cannot be observed with stained or fixed specimens.

Dark field Microscopy 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 Dark field Microscopy a little time to unfold and mature. However, before doing so, let me simply make a couple of comments:

Dark field Microscopy 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.

Dark field Microscopy benefit

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Bright Field vs. Dark Field microscopy

Bright Field vs. Dark Field microscopy

working principle of dark field microscopy do you know what it?


How Dark field Microscopy work?-Microscopes Dark field Microscopy are used to magnify objects. Through magnification, an image is made to appear larger than the original object. The magnification of an object can be calculated roughly by multiplying the magnification of the objective lens times the magnification of the ocular lens. Objects are magnified to be able to see small details. There is no limit to the magnification that can be achieved; however, there is a magnification beyond which detail does not become clearer. The result is called empty magnification when objects are made bigger but their details do not become clearer. Therefore, not only magnification but resolution is important to the quality of the information in an image.

Dark field Microscopy The resolving power of the microscope is defined as the ability to distinguish two points apart from each other. The resolution of a microscope is dependent on a number of factors in its construction. There is also an inherent theoretical limit to resolution imposed by the wavelength of visible light (400-600nm). The theoretical limit of resolution (the smallest distance able to be seen between two points) is calculated as:

Resolution = 0.61 l/N.A.

what is dark field microscopy

where l represents the wavelength of light used and N.A.is the numerical aperture. The student-grade microscopes generally have much lower resolution than the theoretical limit because of lower quality lenses and illumination systems.

Dark field Microscopy Standard brightfield microscopy relies upon light from the lamp source being gathered by the substage condenser and shaped into a cone whose apex is focused at the plane of the specimen. Specimens are seen because of their ability to change the speed and the path of the light passing through them. This ability is dependent upon the refractive index and the opacity of the specimen. To see a specimen in a brightfield microscope, the light rays passing through it must be changed sufficiently to be able to interfere with each other which produces contrast (differences in light intensities) and, thereby, build an image. If the specimen has a refractive index too similar to the surrounding medium between the microscope stage and the objective lens, it will not be seen. To visualize biological materials well, the materials must have this inherent contrast caused by the proper refractive indices or be artificially stained. These limitations require instructors to find naturally high contrast materials or to enhance contrast by staining them which often requires killing them. Adequately visualizing transparent living materials or thin unstained specimens is not possible with a brightfield microscope.

Dark field 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 (Fig. 1). The entire field appears dark when there is no sample on the microscope stage; thus the name Dark field 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 Dark field 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.

The Dark field Microscopy highest quality darkfield microscopes are equipped with specialized costly condensers constructed only for darkfield application. This darkfield effect can be achieved in a brightfield microscope, however, by the addition of a simple “stop”. The stop is a piece of opaque material placed below the substage condenser; it blocks out the center of the beam of light coming from the base of the microscope and forms the hollow cone of light needed for darkfield illumination.

Where need 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)



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