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How to dark field microscopy blood without ruining anyone’s life. (Or your own.)

dark field microscopy blood

When to Use a dark field microscopy blood?

Dark field microscopes are used in a number of different ways to view a variety of specimens that are hard to see in a light field unit. Live bacteria, for example, are best viewed with this type of microscope, as these organisms are very transparent when unstained.

There are multitudes of other ways to use dark field illumination, often when the specimen is clear or translucent. Some examples:

Dark field illumination of caffeine crystalsLiving or lightly stained transparent specimens
Single-celled organisms
Live blood samples
Aquatic environment samples (from seawater to pond water)
Living bacteria
Hay or soil samples
Pollen samples
Certain molecules such as caffeine crystals (right)
Dark field microscopy makes many invisible specimens appear visible. Most of the time the specimens invisible to bright field illumination are living, so you can see how important it is to bring them into view!

dark field microscopy blood

What Disadvantages of dark field microscopy blood?

What Disadvantages of dark field microscopy blood?

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.

dark field microscopy blood

What is dark field microscopy blood?

What is dark field microscopy blood?

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 darkfield illumination.

Darkfield illumination 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, darkfield illumination 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.

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 darkfield illumination, 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. The resolving power of the

dark field microscopy blood

What dark field microscopy blood 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.

dark field microscopy blood 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 syphilis. A newer algorithm that is gaining in popularity begins with a treponemal enzyme immunoassay as the initial test, followed by a non-treponemal test, and if necessary, a “tie-breaker” third test, using a different treponemal test.

dark field microscopy blood

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.

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. dark field microscopy blood 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.

dark field microscopy blood

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