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What is dark field microscopy?

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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.

dark field microscopy live blood analysis

dark field microscopy live blood analysis

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.

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Why 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 is Dark field illumination Applications ?

Viewing Blood cells.
Viewing bacteria.
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 Blood and What Does it Do

Two types of blood vessels carry blood throughout our bodies: The arteries carry oxygenated blood (blood that has received oxygen from the lungs) from the heart to the rest of the body.

The blood then travels through the veins back to the heart and lungs, where it receives more oxygen. As the heart beats, you can feel blood traveling through the body at your pulse points – like the neck and the wrist – where large, blood-filled arteries run close to the surface of the skin.

The blood that flows through this network of veins and arteries is called whole blood. Whole blood contains three types of blood cells:

Red Blood Cells
White Blood Cells

These blood cells are mostly manufactured in the bone marrow (the soft tissue inside our bones), especially in the bone marrow of the vertebrae (the bones that make up the spine), ribs, pelvis, skull, and sternum (breastbone). These cells travel through the circulatory system suspended in a yellowish fluid called plasma (pronounced: plaz-muh). Plasma is 90% water and contains nutrients, proteins, hormones, and waste products. Whole blood is a mixture of blood cells and plasma.
Red Blood Cells

Red blood cells (RBCs, and also called erythrocytes, pronounced: ih-rith-ruh-sytes) are shaped like slightly indented, flattened disks. Red blood cells contain an iron-rich protein called hemoglobin (pronounced: hee-muh-glow-bun). Blood gets its bright red color when the hemoglobin in RBCs picks up oxygen in the lungs. As the blood travels through the body, the hemoglobin releases oxygen to the tissues. The body contains more RBCs than any other type of cell, and each has a life span of about 4 months. Each day, the body produces new RBCs to replace those that die or are lost from the body.

White Blood Cells

White blood cells (WBCs, and also called leukocytes, pronounced: loo-kuh-sytes) are a key part of the body’s system for defending itself against infection. They can move in and out of the bloodstream to reach affected tissues. The blood contains far fewer white blood cells than red cells, although the body can increase production of WBCs to fight infection. There are several types of white blood cells, and their life spans vary from a few days to months. New cells are constantly being formed in the bone marrow.

Several different parts of blood are involved in fighting infection. White blood cells called granulocytes (pronounced: gran-yuh-low-sytes) and lymphocytes (pronounced: lim-fuh-sytes) travel along the walls of blood vessels. They fight germs such as bacteria and viruses and may also attempt to destroy cells that have become infected or have changed into cancer cells.

Certain types of WBCs produce antibodies, special proteins that recognize foreign materials and help the body destroy or neutralize them. Someone with an infection will often have a higher white cell count than when he or she is well because more WBCs are being produced or are entering the bloodstream to battle the infection. After the body has been challenged by some infections, lymphocytes “remember” how to make the specific antibodies that will quickly attack the same germ if it enters the body again.

Platelets (also called thrombocytes, pronounced: throm-buh-sytes) are tiny oval-shaped cells made in the bone marrow. They help in the clotting process. When a blood vessel breaks, platelets gather in the area and help seal off the leak. Platelets survive only about 9 days in the bloodstream and are constantly being replaced by new cells.

Drop of Blood

Blood also contains important proteins called clotting factors, which are critical to the clotting process. Although platelets alone can plug small blood vessel leaks and temporarily stop or slow bleeding, the action of clotting factors is needed to produce a strong, stable clot.

Platelets and clotting factors work together to form solid lumps to seal leaks, wounds, cuts, and scratches and to prevent bleeding inside and on the surfaces of our bodies. The process of clotting is like a puzzle with interlocking parts. When the last part is in place, the clot happens – but if only one piece is missing, the final pieces can’t come together.

When large blood vessels are severed (or cut), the body may not be able to repair itself through clotting alone. In these cases, dressings or stitches are used to help control bleeding.

In addition to the cells and clotting factors, blood contains other important substances, such as nutrients from the food that has been processed by the digestive system. Blood also carries hormones released by the endocrine glands and carries them to the body parts that need them.

dark field microscopy live blood analysis What can test ?

hus, the dark field blood analysis provides information about the function and structure of blood cells and plasma endobionts, as well as developing bacterial and fungal precursors. The dark field examination also indicates changes within the cell through hormonal and mineral deficiencies. It is particularly beneficial for the evaluation of patients with chronic diseases, and children with susceptibility to infection, or recurrent bacterial problems, such as Candida or other fungal diseases. Dark field blood analysis is crucial in answering questions related to any chronic, or toxic problems.

Dark field microscopy is also an important tool in biological therapies. It can be used to test the effect of certain medications, by adding the medication to a blood sample and analyzing the reaction that is produced. This investigation is extremely motivating for the patient, by allowing him to directly experience the diagnosis.

The effects of dark field microscopy can not be replaced by any other blood test, especially not by normal laboratory microscopic blood tests, sent in fixed samples, as the blood changes its function due to environmental changes, so the blood must be tested while it is still fresh. It is also important to evaluate the degeneration tendency of blood samples, especially when considering the behavior of tumors.

The presence of bacteria precursors, which are not disease-inducing, but increase the risk of future disease development, can also be found in the dark field examination. Therefore, this dark field examination of the blood is a valuable and necessary preventative measure.


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