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How to transform your bright field vs dark field tem into a bright field vs dark field tem.

bright field vs dark field tem

How bright field vs dark field tem work?

 

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. The student-grade microscopes generally have much lower resolution than the theoretical limit because of lower quality lenses and illumination systems.

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 instructors to find naturally high contrast materials or to enhance contrast by staining them which often requires killing them. 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 (Fig. 1). The entire field appears dark when there is no sample on the microscope stage; thus the name bright field vs dark field tem. 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 (note the rays scattered by the specimen in Figure 1). 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 bright field vs dark field tem 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.

The highest quality darkfield microscopes are equipped with specialized costly condensers constructed only for darkfield application. This darkfield effect can be achieved in a brightfield microscope, however, by the addition of a simple “stop”. The stop is a piece of opaque material placed below the substage condenser; it blocks out the center of the beam of light coming from the base of the microscope and forms the hollow cone of light needed for darkfield illumination.

bright field vs dark field tem

When to Use a Dark Field Microscope?

bright field vs dark field tem are used in a number of different ways to view a variety of specimens that are hard to see in a light field unit. Live bacteria, for example, are best viewed with this type of microscope, as these organisms are very transparent when unstained.

There are multitudes of other ways to use dark field illumination, often when the specimen is clear or translucent. Some examples:

Dark field illumination of caffeine crystalsLiving or lightly stained transparent specimens

Single-celled organisms

Live blood samples

Aquatic environment samples (from seawater to pond water)

Living bacteria

Hay or soil samples

Pollen samples

Certain molecules such as caffeine crystals (right)

bright field vs dark field tem makes many invisible specimens appear visible. Most of the time the specimens invisible to bright field illumination are living, so you can see how important it is to bring them into view!

bright field vs dark field tem

What is Disadvantages of bright field vs dark field tem?

A dark field microscope can result in beautiful and amazing images; this technique also comes with a number of disadvantages.

First, dark field images are prone to degradation, distortion and inaccuracies.
A specimen that is not thin enough or its density differs across the slide, may appear to have artifacts throughout the image.
The preparation and quality of the slides can grossly affect the contrast and accuracy of a dark field image.
You need to take special care that the slide, stage, nose and light source are free from small particles such as dust, as these will appear as part of the image.
Similarly, if you need to use oil or water on the condenser and/or slide, it is almost impossible to avoid all air bubbles.
These liquid bubbles will cause images degradation, flare and distortion and even decrease the contrast and details of the specimen.
Dark field needs an intense amount of light to work. This, coupled with the fact that it relies exclusively on scattered light rays, can cause glare and distortion.
It is not a reliable tool to obtain accurate measurements of specimens.
Finally, numerous problems can arise when adapting and using a dark field microscope. The amount and intensity of light, the position, size and placement of the condenser and stop need to be correct to avoid any aberrations.

Dark field has many applications and is a wonderful observation tool, especially when used in conjunction with other techniques.

However, when employing this technique as part of a research study, you need to take into consideration the limitations and knowledge of possible unwanted artifacts.

bright field vs dark field tem

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