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difference between bright field microscopy and dark field microscopy

What is  bright field microscopy?

bright field vs dark field microscopy

bright field vs dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

bright field vs dark field microscopy

bright field vs dark field microscopy

What is Bright field 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.

brightfield and darkfield microscopy
difference between bright field microscopy and dark field microscopy
difference between brightfield and darkfield microscopy
differences between bright field vs dark field microscopy
difference between dark field microscopy and brightfield microscope
differentiate between bright field vs dark field microscopy

Bright field 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.

Bright field 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.

Bright field microscopy Advantages

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

Bright field 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 bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

Bright field 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.

What is dark field microscopy?

How darkfield microscopy 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 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 (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 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.

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.

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

difference between bright field microscopy and dark field microscopy

Difference between Dark and Bright Field Illumination –-When you view a particular specimen under a bright field microscope, you will observe that the specimen is dark while its background is bright; hence the name bright field microscope.
On the other hand, when you view a particular specimen under a dark field microscope, you will observe that the specimen is bright while its background is dark; hence the name dark field microscope.
-Since little light actually falls on the specimen, dark-field illumination shows less detail overall than bright-field illumination.

Bright Field Illumination.
A way of illuminating a specimen in a microscope by lighting it from behind, making the specimen appear dark against a bright background. It is considered the most basic type of microscope

The Dark field illumination requires blocking out the central light waves along the optical axis of the light waves. Blocking the light waves allows you to see the specimine when only the oblique rays hit the specimen at an angle.
Parts of the Microscope.
Monitor: To display the picture of the specimen your CMO objective lens is focused on.

CMO Objective lens: To magnify on the part of a specimen you wish to observe.

Stage: To hold your specimen.

Lamp Voltage: Controls how bright the light is.

Zoom Body: To zoom in on the specimen.

Illumination section: Illuminates the specimen.

Brightfield/Darkfield Diascopic Stand. Holds the inner pieces of the microscope inside.

Camera Control: Controls where the CMO Objective lens focuses on.

Economic observation tube: Allows you to see the specimen without the monitor.

Digital camera: Transfers the pixels onto the moniter so you can see the specimen.

Stage: Holds the microscope together and supports the microsope.

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