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Live Cell Analysis

As a preface to this section I wish to make it clearly known that I am aware that this is a very controversial topic. I make no claims to diagnose a disease state or prescribe a treatment solely from this type of study. It is however very interesting to study live cells and valuble information can be obtained through the careful examination of cells in a living environment. I simply consider this one more tool in the tool-box of evaluating a person as a whole. You simply cannot have too many tools in the tool box!


Phase Contrast / Dark Field
Microscopy for Live Cell Studies
Click Here to View Video
of Live Cells Taken
Through a Microscope

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.

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 naturally high contrast materials or to enhance contrast by staining them which often requires killing the biological material. 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. 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. 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.

How is this accomplished in the microscope. The image below shows the setup of the dark phase optical path.

(Redrawn from Gray)*

By the use of a annular stop in the condenser and a phase plate within an objective lens aligned with the annular stop, a light beam can be split and each of the separated beams will pass through the same transparent medium. The light passes through the annular stop and forms a cone of light which comes to its vertex at the focal point of the specimen. Any background light which is not deviated by the specimen goes through the phase ring in the phase plate and is advanced about a quarter of a wavelength. Deviated light passing through the specimen is retarded by about a quarter of a wavelength and passes through the phase plate without going through the ring. When the beams are recombined further along the light path, the differences in the phase of the deviated and undeviated light beams become additive and subtractive. The resultant wave is sum of the two waves which have their crests and troughs opposite each other and is four times darker than the background. Therefore, the specimen appears four times darker than the background. The net result is that features of the object are either lighter or darker than the surrounding field.

Dark phase is the most common optical method used for viewing living cells. All of the inverted tissue culture microscopes employ it for quick examination of cells in tissue culture.

*Diagram redrawn from Gray, P. 1964. Handbook of Basic Microtechnique. McGraw-Hill: New York.

See examples below of Live Cells that are
viewable with this technique.
 
Photos Taken With a Dark Field Microscope

Normal Red Blood Cell (RBCs)
The circulatory system is the means by which oxygen, nutrients, antibodies, and hormones are transported to the cells to keep them alive and functioning. This is how our blood looks when we are experiencing optimum health. The Erythrocytes (cells) are round and separated and move through the capillaries very easily. The average size of healthy RBCs is 7.2 microns
 
Protein Linkage
This condition is the first sign of cell stickiness and may progress into rouleau if not corrected. Protein linkage is a sign that excessive protein is being consumed or the protein is not being digested completely. As the cells start sticking together it becomes harder for the heart to push the blood through the veins and arteries.
 
Rouleau
When the blood gets to this condition the amount of oxygen that can be transported is severely diminished. This condition is caused by high fat and protein diets and high acidity. Your blood will look like this if you drink one soda and will stay that way for at least two hours. Because your cells are not getting oxygen you often feel fatigued, have poor digestion, and skin disorders.
 
Erythrocyte Aggregation
This condition is one step worse than rouleau. This is often seen in people with degenerative diseases. This is caused by undigested fats and proteins and high acidity. Degeneration of tissue always follows low oxygen and acidity. This condition can precede a blood clot which can cause a stroke or heart attack.
 
Poikilocytosis
This condition is caused by free radicals. This also lowers the bloods oxygen carrying capacity and shortens the life if the cell. RBCs don't have nuclei, so they will not mutate, but the fact that there is free radical damage signifies that there will also be damage to the nuclei of tissue cells which is the beginning of mutations that lead to cancer.
 
Microcyte
These are small RBCs having a diameter of less than 5 microns. These cells have less hemoglobin than normal cells and is often seen in people with iron deficiency anemia.
 
Macrocyte
These cells are greater than 10 microns in size. Macrocytes are often seen in people with hemolytic anemia.
 
Anisocytosis
In this condition there are variations in the size of the cells. This is mostly seen in people with low levels of the vitamins B12 and folic acid and the mineral iron.
Target Cell
These RBCs are deficient in iron and therefore hemoglobin, which is the part that carries oxygen. The symptoms produced in the body are tiredness, poor digestion, and anemia
 
Hemolysis
This is literally the destruction of the red blood cell. This can be caused by bacterial infection or any number of toxins introduced into the blood stream. The hemoglobin goes out of the cells interior and diffuses into the plasma. This can also be caused by any hypotonic fluid injected intravenously. Death will follow if not corrected.
 
Fat, Protein, and Liver Congestion

Normal Thrombocytes (Platelets)
Platelets are small disk shaped components of blood that have an important role in blood coagulation. When a blood vessel is injured the platelets adhere to each other and to the injury to form a plug which stops the bleeding. Platelets number approximately 200,000 to 300,000/cu.mm.
Thrombocyte Aggregation
When the thrombocytes (platelets) aggregate when there is no injury a very dangerous situation develops. The aggregated platelets can form a clot which can block an artery causing a stroke or heart attack. This clot is called a thrombus. Diets high in fats and proteins or high sugar consumption can create this situation.
Spicules (Fibrin)
Fibrin are platelets that have changed in shape that form a net-like substance in which blood clots are formed by the entrapment of red and white cells and platelets. When they are formed in the blood when there is no injury there is a risk of a blood clotting that can lead to a heart attack or stroke. This is caused by liver stress due to incomplete digestion of proteins and fats.
Chylous
Chylomicrons are small particles of fats in the blood after the digestion and assimilation of fat in food. The presence of chyomicrons in the blood after a 12 hour fast indicates a condition known as hyperlipo-proteinemia, This can lead to atherosclerosis, coronary artery disease, and enlargement of the liver and spleen.
Plaque
Atherosclerotic plaque is one of the most dangerous conditions in the blood. It can adhere to the artery walls narrowing and hardening them. These crystals are formed when the system becomes acidic and the fatty acids from simple carbohydrates crystallize.
Uric Acid Crystal
Uric acid is a byproduct of protein metabolization and urea. When the body becomes acidic the urea forms crystals that can lodge in the joints or in the tissues. Uric acid is the cause of gout and one of the causes of fibromyalgia. These crystals are shaped like knives and are the reason it can cause so much pain for people with either of these disorders.
Cholesterol Crystal
Cholesterol is an important sterol in the body that is the precursor for many important hormones. Only when the body is acidic does the cholesterol crystallize and become a problem. This is probably the most seen crystal in blood analysis, but it's important to realize that cholesterol is not the problem when you see the crystals, acidity is.
Echinocyte
These are red blood cells that have a thorny appearance. This is usually indicative of kidney stress and crenation (the shrinking of the cell by dehydration).
 
Immune System, Parasites, Bacteria, and Fungal Forms

Healthy White Blood Cell
These cells are made up of lymphocytes and leukocytes. They form the basis of the immune system. There are approximately one or two white cells for every 500 red cells. When there is an elevated count it is usually a sign of an infection. The white blood cells protect us from infectious diseases and will destroy any cells that have mutated.
Yeast
A fungus that feeds on undigested food and sugar in the blood. The principle yeast found in the blood is Candida Albicans. It is usually found in people with cancer, fibromyalgia and those with chronic fatigue. This condition is also indicative of over acidity as yeast cannot live in an alkaline environment.
L-Form Bacteria
This is a bacterial infection (it's hard to see in this picture) that is shaped like a butterfly. This usually signifies a condition of low immunity and high blood sugar.
Rod Form Bacteria
This is an advanced form of a bacterial infection and is regarded as a serious indicator of a weak immune system. These bacteria produce very toxic acid byproducts as a result of their metabolic processes.
Parasitized Red Blood Cells
This is bacteria or parasites that get inside the cells. Of course the cell will die, and unless they are stopped by the immune system, they will continue to attack other cells.
Fungal Forms
Fungi can spread throughout your body through the blood and develop colonies. They usually develop slowly and are hard to diagnose and usually resistant to treatment. They are seldom fatal and most of the times go unnoticed. This is commonly seen in cases of poor assimilation of nutrients and an acidic condition in the body fluids.