Dark field microscopy

Dark field microscopy (Dark Ground Microscopy) is an optical microscopy illumination technique used to enhance the contrast in unstained samples. It works on the principle of illuminating the sample with light that will not be collected by the objective lens, so not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it.

Additional recommended knowledge

Essential Laboratory Skills Guide

Daily Sensitivity Test

Daily Visual Balance Check

The light's path

The steps are illustrated in the figure where an upright microscope is used.  

1. Light enters the microscope for illumination of the sample.
2. A specially sized disc, the patch stop (see figure) blocks some light from the light source, leaving an outer ring of illumination.
3. The condenser lens focuses the light towards the sample.
4. The light enters the sample. Most is directly transmitted, while some is scattered from the sample.
5. 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).
6. 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 are impressive.

The main limitation of dark field microscopy is the low light levels seen in the final image. This means the sample must be very strongly illuminated, and 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 images must be done with great care as common dark features of bright field microscopy images may be invisible, and vice versa.

Dark Field Microscopy used for Live Blood Analysis and its Credibility

Biggest disadvantage of Live Blood Analysis in Dark Field is the fact it is based on observations and interpretations with no general scientific acceptance. Anybody with possibilities of self-publication has therefore the chance to voice his own interpretations. Thus was the situation over 50 years ago already and it will never change.

However, practitioners doing Live Blood Analysis should at least be able to identify blood parts correctly, which are also seen by hematologists in stained bright field samples. This is a must in order to gain any form of credibility of Live Blood Analysis in Dark Field.

Red Blood Cells / Erythrocytes

Erythrocytes are not always the same in form and size. This is visible in stained bright field as well as in live blood dark field samples. Therefore scientifically acknowledged terminology exists for these cells seen in blood.

Anisocytosis, Acanthocytes, Echinocytes, Elliptocytes, Poikilocytes, Reticulocytes, Schistocytes, Stomatocytes, Dakrocytes and Target Cells are hematological terms, which live blood analysts need to know and they should be able to identify those in dark field.

Rouleaux / Coin roll formation

Coin roll formation in dark field is nowadays world-wide abused by sellers of remedies and gadgets to demonstrate bad blood circulation. An absolute favorite is a dark field picture with coin rolls titled “before” and a normal dark field picture titled “after”. Worst of it all, most of times it's not even true rouleaux but blood sludge (thick blood drop).

According to the “Oxford Textbook of Medicine” real rouleaux in a blood sample resembles the visual analogue of a high ESR (erythrocyte sedimentation rate). It is therefore a non-specific indicator of disease.

White Blood Cells / Leucocytes

There are 5 different forms of white blood cells, Neutrophil, Eosinophil and Basophil Granulocytes as well as Monocytes and Lymphocytes. Lymphocytes are divided into B- and T-Lymphocytes and Natural Killer cells (NK). Again, dark field analysts should be able to classify them correctly.

Thrombocytes / Platelets

Platelets are also visible in stained bright field samples. However, Platelet aggregations are only visible in untreated blood smears. The different staining methods separate the Platelet aggregations.

Platelet aggregation, often entangled with fibrin, is frequently observed in dark field. No surprises here - Cardio-vascular diseases are the most prevalent ones these days in the western world. Millions of people world-wide are on Aspirin. It blocks the formation of thromboxane A2 in platelets, producing an inhibitory effect on platelet aggregation. However, rebound effects after cessation have been shown in several studies.

It is amazing how many dark field analysts world-wide diagnose these platelet aggregations as “Candida”. People with systemic candida infections are very ill and usually need treatment in an intensive care unit. They are hardly able to see a physician for live blood analysis in dark field. Candida needs an acidic environment for existence (e.g. vagina pH2).

Normal blood plasma pH is 7.38 – 7.42. If this pH drops below 7.36 metabolic acidosis is imminent.

There are 3 mechanisms for the body to regulate the blood plasma pH: 1. Physiological buffer mechanism 2. Kidney excretion 3. Acid dump in tissues in exchange for alkaline minerals

Even the blood drop of a cancer patient at the time of sampling has a pH of 7.36 or above. Erythrocytes transport oxygen but don’t use it. They have an anaerobic cell metabolism with an end product of lactic acid. Mucor racemosus also produces lactate. Aspergillus niger produces citric and oxalic acids. This leads to a gradual left shift of the pH of live blood samples and allows the pathological developments as described by Professor Enderlein.

Conclusion: The unhealthier a person the quicker the pathological developments.

Interpretation and naming of the pathological developments in live blood samples is a free for all for dark field analysts and frowned upon by Hematologists. However, the credibility of live blood analysis in dark field suffers the most when practitioners are unable to identify blood particles correctly, which can also be seen by Hematologists.

When it comes to the interpretation of developments seen in live blood samples in dark field, nothing beats experience. Professor Enderlein certainly had plenty of this. Over six decades of observing live blood samples in dark field. And remember, he never set out to proof anything with dark field microscopy – he simply recorded everything that he observed meticulously. His conclusion: Only changes in milieu (biological terrain) allows upwards or downwards developments!

Further reading at: www.darkfieldtraining.com [1]

External links and references

http://www.darkfieldtraining.com http://www.microscopyu.com/articles/stereomicroscopy/stereodarkfield.html

• Live blood photos under a Darkfield Microscope

Molecular Expressions: Darkfield Illumination Primer