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difference between dark field and phase contrast microscopy

What is difference between dark field and phase contrast microscopy?

What is difference between dark field and phase contrast microscopy?

Bright-field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample. Bright-field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes, and its simplicity makes it a popular technique. The typical appearance of a bright-field microscopy image is a dark sample on a bright background, hence the name.

difference between dark field and phase contrast microscopy Light path

The light path of a bright-field microscope is extremely simple, no additional components are required beyond the normal light-microscope setup. The light path therefore consists of:

a transillumination light source, commonly a halogen lamp in the microscope stand;
a condenser lens, which focuses light from the light source onto the sample;
an objective lens, which collects light from the sample and magnifies the image;
oculars and/or a camera to view the sample image.

Bright-field microscopy may use critical or Köhler illumination to illuminate the sample.

difference between dark field and phase contrast microscopy Performance

Bright-field microscopy typically has low contrast with most biological samples, as few absorb light to a great extent. Staining is often required to increase contrast, which prevents use on live cells in many situations. Bright-field illumination is useful for samples that have an intrinsic color, for example chloroplasts in plant cells.Bright-field microscopy is a standard light-microscopy technique, and therefore magnification is limited by the resolving power possible with the wavelength of visible light.

difference between dark field and phase contrast microscopy Advantages

Simplicity of setup with only basic equipment required.
Living cells can be seen with bright-field microscopes

difference between dark field and phase contrast microscopy Limitations

Very low contrast of most biological samples.
The practical limit to magnification with a light microscope is around 1300X. Although higher magnifications are possible, it becomes increasingly difficult to maintain image clarity as the magnification increases.
Low apparent optical resolution due to the blur of out-of-focus material.
Samples that are naturally colorless and transparent cannot be seen well, e.g. many types of mammalian cells. These samples often have to be stained before viewing. Samples that do have their own color can be seen without preparation, e.g. the observation of cytoplasmic streaming in Chara cells.

difference between dark field and phase contrast microscopy Enhancements

Reducing or increasing the amount of the light source by the iris diaphragm.
Use of an oil-immersion objective lens and a special immersion oil placed on a glass cover over the specimen. Immersion oil has the same refraction as glass and improves the resolution of the observed specimen.
Use of sample-staining methods for use in microbiology, such as simple stains (methylene blue, safranin, crystal violet) and differential stains (negative stains, flagellar stains, endospore stains).
Use of a colored (usually blue) or polarizing filter on the light source to highlight features not visible under white light. The use of filters is especially useful with mineral samples.

difference between dark field and phase contrast microscopy

difference between dark field and phase contrast microscopy APPLICATIONS

 

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

difference between dark field and phase contrast microscopy

What Advantages and Disadvantages about difference between dark field and phase contrast microscopy?

What Advantages and Disadvantages about difference between dark field and phase contrast microscopy?

No one system is perfect, and dark field microscopy may or may not appeal to you depending on your needs.

Some advantages of using a dark field microscope are:

Extremely simple to use
Inexpensive to set up (instructions on how to make your own dark field microscope are below)
Very effective in showing the details of live and unstained samples
Some of the disadvantages are:

Limited colors (certain colors will appear, but they’re less accurate and most images will be just black and white)
Images can be difficult to interpret to those unfamiliar with dark field microscopy
Although surface details can be very apparent, the internal details of a specimen often don’t stand out as much with a dark field setup.

Below are contrasting examples of dark field (left) versus bright field (right) illumination of lens tissue paper. Note how they both create a different style of image.

Dark field illumination Bright field illumination

Admit it, by now you’re curious to check out your own dark field! You can create one with minimal time and effort. Just read on…

difference between dark field and phase contrast microscopy

What Makes Up Healthy Blood?

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.

(Live Blood Examination in the Darkfield according to Prof. Dr. G. Enderlein)
difference between dark field and phase contrast microscopy or Live Blood Analysis is a way of studying live whole blood cells under a specially adapted microscope that projects the dynamic image onto a video screen. This allows you to view your inner terrain. Digestive, eliminative and immune functions can be assessed as well as the presence of bacteria and other micro-organisms.

The darkfield microscopic examination of the freshly taken live blood is one of the most important examinations of the holistic medicine applied at the Centre. It enables us to view the inner terrain (milieu) and to examine the functions of the red blood cells. It also shows the evolutionary stages of the smallest proteins (endobionts) which are found in every human body. We are also able to see any developed structures such as bacteria, virus and fungus. The darkfield examination shows the state of the blood cells, endobionts and the plasma in a functional and structural way, making bacterial processes and fungal pre-stages in the blood clearly visible.

The darkfield examination is most suitable for the evaluation of chronic diseases; for children who are prone to infections; for recurrent bacterial problems; for candida and other fungal problems and also to answer questions concerning chronic problems of toxicity (e.g. amalgam disturbances).

difference between dark field and phase contrast microscopy

What is difference between dark field and phase contrast microscopy?

Similar to a bright field but it is modified by a dark field stop just below the source. The dark field stop is just  the condenser, and blocks the light in the center of the lightsource so that the only light that goes through is around the edges. That light is then bent by the condenser and diffacts off the specimen. None of the light goes directly from the light source into the objective, so if there is no specimen, the image will be very dark. The specimen in this method will be illuminated against a black background.
A dark field microscope is useful because it increases the contrast of the image and does not use stains. The lack of staining means that it can be used on live specimens and that one can observe the motility of the organism as well as its correct morphology. Usually, the stains and enzymes used in labs can distort the shape of the organism, but that isn’t an issue with dark field microscopy. This method can also be used to see organisms that are hard to stain, such as Treponema pallidum, spirochetes, and mycoplasma.The one downside is that it’s not possible to see the inclusions, or internal details of the cell

difference between dark field and phase contrast microscopy

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