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dark field microscopy principle

What is dark field microscopy principle?

What is dark field microscopy principle?

dark field microscopy of sugar crystals
Dark Field illumination is a technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.

Pictured right: Highly magnified image of sugar crystals using darkfield microscopy technique

When light hits an object, rays are scattered in all azimuths or directions. The design of the dark field microscope is such that it removes the dispersed light, or zeroth order, so that only the scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.

dark field microscopy principle

What is dark field microscopy principle?

What is dark field microscopy principle?

Dark Field microscopy is a microscope illumination technique used to observe unstained samples causing them to appear brightly lit against a dark, almost purely black, background.

When light hits an object, rays are scattered in all directions. The design of the dark field microscope is such that it removes the dispersed light so that only the scattered beams hit the sample.

The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or refracted off the object, ultimately, allowing you to view a specimen in dark field.

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.

Dark field can be used to study marine organisms such as algae and plankton, diatoms, insects, fibres, hairs, yeast, live bacterium, protozoa as well as cells and tissues and is ideal for live blood analysis enabling the practitioner to see much more than is possible with other lighting methods.

dark field microscopy principle

dark field microscopy principle principle

dark field microscopy principle principle

dark field microscopy principle is a method which also creates contrast between the object and the surrounding field. As the name implies, the background is dark and the object is bright. A annular stop is also used for dark field, but the stop is now outside the field of view. Only light coming from the outside of the beam passes through the object and it cannot be seen directly. Only when light from the stop is deflected and deviated by the object can it be seen. This method also produces a great deal of glare and therefore the specimen often appears as a bright silhouette rather than as a bright object of which much detail can be determined. The following diagram shows the setup of the dark field light path.

dark field microscopy principle

dark field microscopy principle syphilis how to check it?

dark field microscopy principle syphilis how to check it?

Darkfield microscopy

In 1830, J.J. Lister (the father of Joseph Lister) invented the darkfield microscope, in which the standard brightfield (Abbe) condenser is replaced with a single- or double-reflecting darkfield condenser. The use of indirect light allows visualization of organisms too small to be seen under direct-light microscopy. In 1906 in Vienna, Karl Landsteiner and Viktor Mucha were the first to use darkfield microscopy to visualize T pallidum from syphilis lesions. Since then, darkfield microscopy has served a vital role in the diagnosis of infectious syphilis.

Clinicians and laboratorians should use universal precautions in collecting, transporting, and handling specimens for darkfield examination. Acquisition of syphilis through occupational exposures, including contact with specimens collected for darkfield microscopy, has been reported.

Proper specimen collection and handling is critical for optimizing the sensitivity of darkfield testing. The clinician should gently cleanse and abrade the lesion with moist gauze, while trying not to cause bleeding. The goal is to obtain serous exudate, while minimizing contamination by blood or pus caused by secondary infection. The clinician might need to apply pressure at the margins of the lesion to express adequate serous fluid. The clinician transfers the serous fluid to a glass slide, either by direct application of the slide to the lesion, or by transferring the fluid with a bacteriologic loop or the edge of a cover slip. If necessary to prevent drying of the specimen, a drop of non-bacteriostatic normal saline may be placed on the slide; however, the saline might dilute the specimen and reduce test sensitivity. The clinician places a cover slip on top of the specimen. A trained microscopist then examines the specimen as soon as possible, no greater than 20 minutes after specimen collection. Placing the slide in a closed container such as a Petri dish during transport to the microscope might reduce evaporative drying.

Definitive identification of T pallidum depends on visualizing not only its typical morphology but also its typical motility. T pallidum is a delicate, tightly spiraled, corkscrew-shaped organism that rotates as it slowly moves backwards and forwards (translational movement); these movements are sometimes accompanied by a slight side-to-side oscillation. T pallidum will occasionally flex or bend sharply in the middle when obstructed by cellular elements or debris in the field but then spring back to its usual linear shape. In the genital region, Treponema refringens, which is part of the normal genital flora, can be distinguished from T pallidum by T refringens’ more coarsely wound spirals, greater flexibility, and rapid translational movement across the slide. In addition, the less experienced observer must guard against misidentifying Brownian movement of fibers or other linear debris as T pallidum.

After a methodical scanning of the entire specimen field of each slide, results are reported as one of the following:

Positive darkfield: Organisms with the characteristic morphology and motility of T pallidum observed

Negative darkfield: Either no treponemes found or spiral organisms seen but without the characteristics of T pallidum.

Unsatisfactory darkfield: The specimen could not be interpreted either due to drying or the presence of too many refractile elements, such as blood cells or fibers.Diagnosis and Management of Syphilis
Darkfield microscopy for point-of-care syphilis diagnosis
syphilis is a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum subspecies pallidum. Globally, an estimated 12 million cases of syphilis occur annually. In the United States, 13,997 cases of primary and secondary (infectious) syphilis were reported to the Centers for Disease Control and Prevention (CDC) in 2009, a 3.7% increase from 2008 and a 134% increase from 2000, when a post-war low of 5,979 primary and secondary syphilis cases was reported. Men who have sex with men (MSM) — especially those who are HIV infected — and blacks are disproportionately affected by syphilis. Geographically, urban areas and the Southeastern region of the United States have the highest rates.

Syphilis is most commonly transmitted by skin-to-skin (or mucous membrane) contact. Following exposure, the infection passes through the following stages:

Primary syphilis, characterized by a painless ulcer, called a chancre, usually develops three weeks after exposure (range 10 days to 90 days) at the site of inoculation. The chancre heals spontaneously after several weeks.

Secondary syphilis is most often characterized by a generalized rash that also resolves without treatment. Rash on the palms and soles can also occur, as can systemic manifestations such as fever, malaise, and lymphadenopathy. Given the widely variable nature of the rash and other manifestations of the disease, syphilis has acquired the moniker “The Great Imitator.”

Early (one year) latent syphilis, defined by the absence of signs or symptoms of disease and diagnosed by serologic evidence of infection.

Tertiary syphilis, which affects about a third of untreated patients and manifests with cutaneous, cardiovascular, or neurologic disease.

Syphilis can also be acquired in utero at any stage of pregnancy and lead to congenital syphilis. Routine syphilis screening and treatment in pregnant women has made congenital syphilis rare in the United States.

Approaches to syphilis diagnosis

Because T pallidum is too fragile an organism to be cultured in the clinical setting, diagnostic testing relies on two approaches: direct detection of the organism and indirect evidence of infection.

Syphilis – Treponema pallidum on darkfield.
Direct methods include darkfield microscopy, molecular assays to detect T pallidum DNA, and histopathologic examination of biopsies of skin or mucous membranes (which can also provide indirect evidence of infection, on the basis of patterns of inflammation in the tissue). Direct methods have the advantage, in some cases, of detecting infection before a patient has mounted a measurable antibody response that results in a reactive serologic test result.

Darkfield microscopy allows visualization of live treponemes obtained from a variety of cutaneous or mucous membrane lesions, as follows.

In primary syphilis, the chancre teems with treponemes that can be seen with darkfield microscopy. The sensitivity of darkfield microscopy for the diagnosis of primary syphilis is approximately 80%. Darkfield sensitivity declines over time and can also decrease if the patient has applied topical antibiotics to the lesion(s). Of note, the mouth harbors normal non-pathogenic treponemes that are indistinguishable microscopically from T pallidum. Therefore, oral specimens cannot be used for darkfield microscopy because of the possibility of false-positive test results.

In secondary syphilis, mucous patches (as long as not oral) and condyloma lata (found in moist areas between body folds) are appropriate specimens for darkfield microscopy. Dry skin lesions usually do not contain sufficient organisms for darkfield testing.

In congenital syphilis, moist discharge from the nose (snuffles) and vesiculobullous lesions of the skin are high-yield specimen sources for darkfield testing.

Indirect methods of diagnosis include serologic testing of blood or cerebrospinal fluid (CSF) and detection of CSF abnormalities (elevated white blood cell count or protein) consistent with neurosyphilis. Serologic testing of blood involves demonstration of host antibody to either endogenous antigens (non-treponemal tests) or to antigens of T pallidum (treponemal tests). Non-treponemal tests, including the rapid plasma reagin test and the venereal disease research laboratory test, have historically been used as the initial screening tests for the serologic diagnosis of syphilis. If a patient’s non-treponemal test is reactive, confirmatory testing with a treponemal test is performed, using either the T pallidum particle agglutination test, the fluorescent treponemal antibody-absorbed test, or another treponemal test. A reactive treponemal test confirms the diagnosis of a new or previously treated case of syphilis. If the treponemal test is non-reactive, the positive non-treponemal test result is considered a biologic false-positive that is not diagnostic of syph

Syphilis is a legally reportable disease in all health jurisdictions in the United States. A positive darkfield examination should trigger a case report, regardless of clinical presentation or serologic results.

Because up to 25% of patients with primary syphilis have non-reactive serologic test results for syphilis, darkfield microscopy provides a critical complementary role in the identification of infectious syphilis. Darkfield microscopy requires, however, a special microscope and a trained microscopist in close proximity to where patients are examined, and few clinical facilities other than STD clinics and some hospitals have the capacity to perform darkfield microscopy. Given the resurgence of syphilis in the United States, the development and maintenance of facilities and skills to perform darkfield microscopy are essential to syphilis prevention and control.

Elaine F. Pierce, MD, MPH, and Kenneth A. Katz, MD, MSc, MSCE, work in the HIV, STD, and Hepatitis Branch of Public Health Services in the Health and Human Services Agency of the County of San Diego in San Diego, CA.

dark field microscopy principle

What is dark field microscopy principle?

What is dark field microscopy principle?

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.

dark field microscopy principle

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