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Bright Field vs. Dark Field microscopy

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What is Darkfield Microscopy?

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.

dark field microscopy ppt-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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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.

dark field microscopy ppt-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

dark field microscopy ppt-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.

dark field microscopy ppt-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:

dark field microscopy ppt-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 microscopy is a specialized illumination technique that capitalizes on oblique illumination to enhance contrast in specimens that are not imaged well under normal brightfield illumination conditions. After the zeroth order (direct) light has been blocked by an opaque stop in the substage condenser, light passing through the specimen from oblique angles at all azimuths is diffracted, refracted, and reflected into the microscope objective to form a bright image of the specimen superimposed onto a dark background.

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dark field microscopy ppt-Transmitted Darkfield Illumination – Transmitted darkfield illumination can be used to increase the visibility of specimens lacking sufficient contrast for satisfactory observation and imaging by ordinary brightfield microscopy techniques. This section discusses various aspects of darkfield illumination, including theory of the technique, condenser design for transmitted darkfield illumination (at both low and high magnifications), microscope configuration parameters, and suggestions for choosing suitable candidates for observation.

dark field microscopy ppt-Reflected Darkfield Illumination – Darkfield illumination with reflected light enables visualization of grain boundaries, surface defects, and other features that are difficult or impossible to detect with brightfield illumination. The technique relies on an opaque occluding disk, which is placed in the path of the light traveling through the vertical illuminator so that only the peripheral rays of light reach the deflecting mirror. These rays are reflected by the mirror and pass through a hollow collar surrounding the objective to illuminate the specimen at highly oblique angles.

Darkfield Illumination for Stereomicroscopy – Darkfield observation in stereomicroscopy requires a specialized stand containing a reflection mirror and light-shielding plate to direct an inverted hollow cone of illumination towards the specimen at oblique angles. A number of aftermarket products are currently available for retrofitting stereomicroscopes with transmitted darkfield illumination. In addition, many of the microscope manufacturers offer illumination accessories that can be conveniently utilized to achieve darkfield conditions for their stereo systems. The principal elements of darkfield illumination are the same for both stereomicroscopes and more conventional compound microscopes.

Darkfield Microscope Configuration – A step-by-step guide to configuration of transmitted light microscopes for use with both low and high magnification darkfield condensers is provided in this review. Careful attention should always be given to microscope alignment and configuration, irrespective of whether the illumination mode is brightfield, darkfield, phase contrast or some other contrast enhancement technique. Time spent in this endeavor will be repaid in excellent performance of the microscope both for routine observation and critical digital imaging or photomicrography.

what is dark field microscopy

dark field microscopy ppt-Troubleshooting Darkfield Microscopy – There are numerous common problems associated with darkfield microscopy and photomicrography or digital imaging. These range from insufficient illumination and condenser mis-alignment to using a field stop of incorrect size. Most darkfield illumination problems are associated with the substage condenser, and this should be the first suspect when things do not work properly. This section addresses some of the more common problems encountered with darkfield microscopy, along with suggested remedies.

Darkfield Photomicrograph Gallery – The Molecular Expressions gallery of darkfield illumination photomicrography and digital imaging contains a wide spectrum of images captured under a variety of conditions and utilizing many different specimens. Included in this unique gallery are specimens ranging from simple diatoms to fossilized dinosaur bones, insects, Moon rocks, and integrated circuits.

Darkfield Microscopy Interactive Tutorials – Explore various aspects of darkfield microscopy theory and practice using these tutorials, which are designed to complement text pages by enabling visitors to use a web browser to simulate configuration and operation of a microscope under darkfield illumination. Both the theory and practice of darkfield microscopy are addressed by the tutorials.


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