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dark field microscopy is primarily used for

What dark field microscopy?

dark field microscopy is primarily used for

dark field microscopy is primarily used for

dark field microscopy is primarily used for

dark field microscopy is primarily used for

Dark field microscopy-dark field microscopy (dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to scatter the beam) is generally dark.

Light microscopy applications

In optical microscopy, dark field microscopy describes an illumination technique used to enhance the contrast in unstained samples. It works by illuminating the sample with light that will not be collected by the objective lens and thus will not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it.

 

The light’s path

The steps are illustrated in the figure where an inverted microscope is used.
Diagram illustrating the light path through a dark field microscopy microscope

Light enters the microscope for illumination of the sample.
A specially sized disc, the patch stop (see figure), blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can also be reasonably substituted at low magnification.
The condenser lens focuses the light towards the sample.
The light enters the sample. Most is directly transmitted, while some is scattered from the sample.
The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct-illumination block (see figure).
Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.

Advantages and disadvantages

dark field microscopy is a very simple yet effective technique and well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-borne, single-celled organisms. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.

The main limitation of dark field microscopy is the low light levels seen in the final image. This means that the sample must be very strongly illuminated, which can cause damage to the sample. dark field microscopy techniques are almost entirely free of artifacts, due to the nature of the process. However, the interpretation of dark field microscopy images must be done with great care, as common dark features of bright-field microscopy images may be invisible, and vice versa.

While the dark field microscopy image may first appear to be a negative of the bright-field image, different effects are visible in each. In bright-field microscopy, features are visible where either a shadow is cast on the surface by the incident light or a part of the surface is less reflective, possibly by the presence of pits or scratches. Raised features that are too smooth to cast shadows will not appear in bright-field images, but the light that reflects off the sides of the feature will be visible in the dark field microscopy images.

Use in computing

dark field microscopy has recently been used in computer mouse pointing devices, in order to allow an optical mouse to work on transparent glass by imaging microscopic flaws and dust on its surface.

dark field microscopy combined with hyperspectral imaging

When coupled to hyperspectral imaging, dark field microscopy becomes a powerful tool for the characterization of nanomaterials embedded in cells. In a recent publication, Patskovsky et al. used this technique to study the attachment of gold nanoparticles (AuNPs) targeting CD44+ cancer cells.

Transmission electron microscope applications

dark field microscopy studies in transmission electron microscopy play a powerful role in the study of crystals and crystal defects, as well as in the imaging of individual atoms.

Conventional dark field microscopy imaging

Briefly, imaging involves tilting the incident illumination until a diffracted, rather than the incident, beam passes through a small objective aperture in the objective lens back focal plane. dark field microscopy images, under these conditions, allow one to map the diffracted intensity coming from a single collection of diffracting planes as a function of projected position on the specimen and as a function of specimen tilt.In single-crystal specimens, single-reflection dark field microscopy images of a specimen tilted just off the Bragg condition allow one to “light up” only those lattice defects, like dislocations or precipitates, that bend a single set of lattice planes in their neighborhood. Analysis of intensities in such images may then be used to estimate the amount of that bending. In polycrystalline specimens, on the other hand, dark field microscopy images serve to light up only that subset of crystals that are Bragg-reflecting at a given orientation.

Weak-beam imaging

Weak-beam imaging involves optics similar to conventional dark field microscopy, but use of a diffracted beam harmonic rather than the diffracted beam itself. Much higher resolution of strained regions around defects can be obtained in this way.

Low- and high-angle annular dark field microscopy imaging

Annular dark field microscopy imaging requires one to form images with electrons diffracted into an annular aperture centered on, but not including, the unscattered beam. For large scattering angles in a scanning transmission electron microscope, this is sometimes called Z-contrast imaging because of the enhanced scattering from high-atomic-number atoms.

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

Digital dark field microscopy analysis

This a mathematical technique intermediate between direct and reciprocal (Fourier-transform) space for exploring images with well-defined periodicities, like electron microscope lattice-fringe images. As with analog dark field microscopy imaging in a transmission electron microscope, it allows one to “light up” those objects in the field of view where periodicities of interest reside. Unlike analog dark field microscopy imaging it may also allow one to map the Fourier-phase of periodicities, and hence phase gradients, which provide quantitative information on vector lattice strain.

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How dark field microscopy work?

Microscopes 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.

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.

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.

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.

Darkfield 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 darkfield 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 darkfield 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 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.

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysisdark field microscopy blood analysis

dark field microscopy blood analysis

How to Make a dark field microscopy

You don’t need to buy a huge expensive set-up to experiment with dark field microscopy.

To create a dark field, an opaque circle called a patchstop is placed in the condenser of the microscope. The patchstop prevents direct light from reaching the objective lens, and the only light that does reach the lens is reflected or refracted by the specimen. Easy enough, right?

If you want to make a dark field microscopy you’ll first need a regular light microscope. Below is your full list of “ingredients”:

dark field microscopyMicroscope
Hole punch
Black construction paper
Transparency film
Glue
Scissors
Pen
Now use the following steps to make your patchstop:

Set up your microscope and choose the lowest-power objective lens.
Set the eyepiece aside somewhere safe.
Open the diaphragm as wide as possible. Then slowly close it until is just encroaches on the circle of visible light.
Now bend over and take a look at the diaphragm from below. See that opening? It’s only slightly smaller than the finished patchstop you’ll create.
Punch a few circles in the black construction paper with the hole punch. Measure one against the diaphragm opening. If it’s more than 10% larger, cut it down to about that size (10% larger than the diaphragm opening). If it’s smaller, cut out a larger circle.
Cut a 5 cm square of transparency paper.
Glue the black circle onto the transparency film, about 2 cm from the corner of the square. In that free 2 cm of paper, write the correct magnification power of your objective.
Mark the patchstop with the correct magnification power.
Repeat the above steps for all the objective powers except the oil immersion lenses.
Now use your patchstop to turn a light field unit into a dark field microscopy:

Select the correct patchstop for the objective power to be used.
Slip the patchstop between the filter holder and condenser. If your microscope has no filter, hold it manually below the condenser.
Remove the eyepiece.
Open the diaphragm and move the patchstop until the light is blocked entirely. Use tape to secure it if there is no condenser on your microscope.
Replace the eyepiece and examine the sample.
As you can see, a dark field microscopy can let users see specimens in a whole new way, bringing those into focus that don’t stand out under intense light. Using dark field microscopy can open up a whole new view of microscopy
The first picture of the plankton was taken by Uwe Kils and is from Wikipedia under the GNU Free Documentation License.

 

dark field microscopy is primarily used for

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)

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.

working principle of dark field microscopy

what is dark field microscopy

image description

dark field microscopy 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.

 

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