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What is dark field microscopy blood analysis?

What is dark field microscopy?

advantages of dark field microscopy

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

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

dark field microscopy blood analysis

What is Dark field microscopy ?-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 microscopy 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.

Have you ever heard of a Dark field microscopy ? While such a name may sound like a sci-fi gadget used to measure black holes, in reality it’s just a handy tool used to view certain types of translucent samples. The average microscope user may not know about the concept of dark field microscopy, yet it can shed new light on the old way of viewing specimens.
Most people who have survived a biology class know what a light field microscope is. This type of scope uses bright field illumination, meaning it floods the specimen with white light from the condenser without any interference. Thus the specimen shows up as a dark image on a light background (or white field if you will).

This type of unit works best with specimens that have natural color pigments. The samples need to be thick enough to absorb the incoming light; so staining is usually paired with this type of microscope.

Plankton illuminated with a Dark field microscopy Yet what if the specimen is light colored or translucent, like the plankton on the right? It certainly won’t stand out against a strong white background. Additionally, some specimens are just too thin. They cannot absorb any of the light that passes through them, so they appear invisible to the user. This is where the concept of dark field illumination comes in!

Rather than using direct light from the condenser, one uses an opaque disk to block the light into just a few scattered beams. Now the background is dark, and the sample reflects the light of the beams only. This results in a light colored specimen against a dark background (dark field), perfect for viewing clear or translucent details.

On a grand scale, the same thing happens every day when you look up at the sky. Do the stars disappear when it’s light out? Of course not! They’re still there, their brilliance blotted out by the mid-day sun.

If you’re still having a hard time visualizing this concept, think of a dusty room with the light on and the door open. You may feel the dust affecting your breathing, but you probably won’t see it flying through the air.

Now turn off the light and close the door to just a sliver, while leaving the light on in the adjacent room. If you look at that sliver of light coming through the door, you’ll see all sorts of dust motes suspended in it. You’re employing a similar principle when you use dark field illumination!

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dark field microscopy blood analysis
dark field microscopy for live 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

dark field microscopy blood analysis

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.

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 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 Dark field microscopy .

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 analysisdark 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

What is Advantages of Dark Field Microscopy?

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.

You can use dark field to study marine organisms such as algae and plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some ceramics.

You can also use dark field in the research of live bacterium, as well as mounted cells and tissues.

It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.

Dark field microscopy is often dismissed for more modern observation techniques such as phase contrast and DIC, which provide more accurate, higher contrasted images and can be used to observe a greater number of specimens.

Recently, dark field has regained some of its popularity when combined with other illumination techniques, such as fluorescence, which widens its possible employment in certain fields.

What Disadvantages of Dark Field Microscopy

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.

 

Where need the dark field microscopy?

Dark field microscopy Applications

Viewing blood cells (biological Dark field microscopy , combined with phase contrast)
Viewing bacteria (biological Dark field microscopy , often combined with phase contrast)
Viewing different types of algae (biological Dark field microscopy )
Viewing hairline metal fractures (metallurgical Dark field microscopy )
Viewing diamonds and other precious stones (gemological microscope or stereo Dark field microscopy )
Viewing shrimp or other invertebrates (stereo Dark field microscopy )

 

What is Dark Field Microscopic Blood Analysis?

You may find it difficult to locate many medical doctors that use this technique. The FDA does not approve of dark field microscopic blood analysis, therefore many doctor’s hands are tied. Viewing a fresh, natural blood sample (a sample not altered with any stains, etc., needed for normal microscopic exams), under the technology of a dark field microscope, will reveal conditions of your blood not normally even considered during the diagnosis of a normal blood test performed in doctor’s office or a lab.

However, an increasing number of health professionals have found that the use of this technique allows inspection of cellular dynamics which as noted above normally escape analysis or diagnosis using orthodox medical tests.

A dark field microscope is a microscope designed to permit diversion of light rays and illumination, from the side, so that details appear light against a dark background; as opposed to light passing straight through the specimen. If bright lights from the microscope pass directly through the specimen, the heat from the light source will kill the red blood cells (RBC)s faster. Also, by diverting the light rays, a greater amount of depth and details can be viewed. (Almost like a three-dimension view).

 

Dark Field Microscopy thus allows a health professional to evaluate the shapes and other properties of individual blood cells, indicating nutritional conditions which can be adversely affecting a person’s health. The advantage of this analysis over standard blood tests, which detect chemical changes in the blood, is the ability of dark field microscopy to detect nutritional disorders sooner, when the problem is in its infancy stages. By monitoring the blood’s condition, a health professional can assist in “balancing” the blood by giving dietary and lifestyle recommendations which can enhance health.

 

This microscopic photograph of healthy, powerful blood shows the red blood cells to be round, evenly shaped and freely floating in plasma. The plasma itself is clear with a few fat globules. There are no signs of clotting, bacteria, fungus, disease or stress. This is the kind of blood a healthy person should have flowing through their circulatory system

 

In darkfield microscopy, one is therefore able to observe “live blood.” Unlike the techniques of electron microscopy, no fixative is used so the picture is one of mobility rather than fixity. With stains and fixatives, the picture reveals a moment in time rather than a continuum.

What one sees in the mobile situation are the usual red blood cells, white blood cells, plasma—and what is floating in the plasma. Microbial activity, undigested food, fungi, and crystals are all apparent as is the capacity of the red blood cells to circulate and the white blood cells to devour morbid matter.

 

As we know, red blood cells transport oxygen to the tissues of the body. Without oxygen, we are devitalized, and according to some theories, the tissues go into a morbid state in which they can survive on fermentation rather than oxygenation. This is what is referred to as anaerobic and it is believed, by such persons as Nobel laureate Prof. Otto Warburg, that cancer thrives in such oxygen deficient conditions.

 

With darkfield microscopy, one often sees sees a condition called “rouleau” in which the red blood cells are stacked together as shown below. Some people believe it is because of the stress on the body of poor metabolism and others believe it is due to this as well as pH (acid-alkaline balance), wrong dietary choices or the presence of excessively high levels of free radicals. In any event, it is usually correctable.

Another condition that is often revealed in these tests is one in which the activity of red blood cells is compromised because of infection, bacterial or viral. In some cases, the red blood cells are misshapen or debilitated by parasitic invasion.

In the photograph above, the “rouleau” effect shows that the red blood cells are clumped together and stacked like coins. Rouleau affects proper oxygenation because the red blood cells do not circulate well enough to deliver oxygen where it is needed.

The condition also favors the growth of unhealthy organisms that can survive in a milieu that is less oxygen rich. Fungi, bacteria, and viruses require less oxygen than healthy tissue.

In the case of rouleau, since oxygenation is really critical to well being, the right diet and herbs may alleviate one of the underlying factors that contributes to cancer. However, enzymes, avoidance of the wrong foods, and protocols that address the specific issues of the patient would be expected to be more effective than more random efforts to ward off ill health.

For instance, one may or may not be iron deficient, but one may have room for improvement in diet and digestion as well as perhaps liver and immune function. Detoxification and decongestion can also be helpful.

 

Typically, a detoxifying herb will also be decongesting and sometimes also somewhat anti-parasitic, but not all herbal alkaloids are the same and not all formulae have the same actions. Therefore consultation with a practitioner who is knowledgeable in the areas that are pertinent is practical and, more importantly, often wise!

 

If the real problem is infection—and devitalization or cancer are secondary to infection—it is important to address the infection so that the red blood cells can “get back to their primary task,” which, of course, is to deliver oxygen to the tissues.

 

The idea that cancer is a disease of degeneration has had its fashionable phases and its days of rejection. The issue of whether an abnormal condition could perpetuate itself in a healthy internal environment, what is called “biological terrain” in the literature, is also debated but not resolved.

 

 

 

 

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