what is darkfield microscope?
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.
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.
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.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.
Brightfield microscopy uses light from the lamp source under the microscope stage to illuminate the specimen. This light is gathered in the condenser, then shaped into a cone where the apex is focused on the plane of the specimen. In order to view a specimen under a brightfield microscope, the light rays that pass through it must be changed enough in order to interfere with each other (or contrast) and therefore, build an image. At times, a specimen will have a refractive index very similar to the surrounding medium between the microscope stage and the objective lens. When this happens, the image can not be seen. In order to visualize these biological materials well, they must have a contrast caused by the proper refractive indices, or be artificially stained. Since staining can kill specimens, there are times when darkfield microscopy is used instead.In darkfield microscopy the condenser is designed to form a hollow cone of light , as apposed to brightfield microscopy that illuminates the sample with a full cone of light. In darkfield microscopy, the objective lens sits in the dark hollow of this cone and light travels around the objective lens, but does not enter the cone shaped area. The entire field of view appears dark when there is no sample on the microscope stage. However, when a sample is placed on the stage it appears bright against a dark background. It is similar to back-lighting an object that may be the same color as the background it sits against – in order to make it stand out.
What darkfield microscope benefit?
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.
Where darkfield microscope application?
Viewing Blood cells.
Viewing different types of algae.
Viewing hairline metal fractures.
Viewing diamonds and other precious stones.
Viewing shrimp and other vertebrae.
Advantages and Disadvantages of Bright Field Microscopy.
Application of Bright Field Illumination
-This technique is widely used in pathology to
view fixed tissue sections or cell films/smears
-In biological applications, brightfield observation is widely used for stained or naturally pigmented or highly contrasted specimens mounted on a glass microscope slide.
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.