The dark field microscope it is a special optical instrument used in certain laboratories. This is the result of a modification made to brightfield microscopy. Dark field microscopy can be achieved by trans-illumination or by epi-illumination.
The first is based on blocking the light rays that reach the condenser directly, through the use of devices that interpose before the light rays reach the condenser..
The dark field with transmitted light makes it possible to highlight the structures, being able to observe extremely thin particles. Structures are seen with some refraction or brightness on a dark background.
While the epi-illumination effect is achieved with incident or oblique light. In this case, the microscope must be equipped with a special filter in the shape of a crescent..
With incident lighting, the observed structures are characterized by presenting a visual effect in high relief. This property makes it possible to highlight the edges of the suspended particles.
Unlike brightfield microscopy, darkfield microscopy is especially useful for visualizing fresh slides containing suspended particles, without any staining..
However, it has several disadvantages, among them that it cannot be used for dry preparations or stained preparations. It does not have a good resolution. Furthermore, to ensure a good image, the numerical aperture of the objectives cannot exceed that of the condenser..
Article index
The composition of the dark-field microscope presents important modifications with respect to the bright-field one, since the fundamentals of both microscopies are opposite..
While in the bright field the light rays are concentrated so that they pass through the sample directly, in the dark field the beams are scattered so that only the oblique beams reach the sample. These are then dispersed by the same sample, transmitting the image towards the objective.
If it were to focus on a slide without a sample, a dark circle would be observed, since without a sample there is nothing to scatter the light towards the objective.
To obtain the desired effect in the visual field, it is necessary to use specific condensers, as well as diaphragms that help control the light beams..
In a dark field field of view the elements or particles in suspension look bright and refractive while the rest of the field is dark, making a perfect contrast.
If oblique or incident light is used, an edge effect with high relief is obtained in the observed structures.
It is the device through which the image reflected and magnified by the objective travels until it reaches the eyepiece or eyepieces.
It is the support where the different objectives are located. The targets are not fixed, they can be removed. The revolver can rotate in such a way that the target can be changed when the operator needs it..
This screw is used to focus the specimen, it moves forward or backward to bring the specimen closer to or farther from the target, and the movement is grotesque..
The micrometer screw is moved forward or backward to move the specimen closer to or farther from the target. The micrometric screw is used for very fine or delicate movements, almost imperceptible. He is the one that achieves the ultimate focus.
It is the support where the specimen will rest on the slide. It has a central opening through which the light beams pass. When the macro and micrometer screws are moved, the stage goes up or down, depending on the movement of the screw.
The carriage allows the entire sample to be traversed with the objective. The allowed movements are forward and backward and vice versa, and from left to right and vice versa.
These are located on the stage, are made of metal and their function is to hold the slide to prevent it from rolling during observation. It is important that the sample remains fixed while it is being observed. Fasteners are exactly sized to receive the slide.
The arm joins the tube with the base. It is the place where the microscope should be held when it is going to be moved from one side to the other. With one hand you take the arm and with the other hand you hold the base.
As its name implies, it is the base or support of the microscope. Thanks to the base the microscope is able to remain fixed and stable on a flat surface.
They are cylindrical in shape. They have a lens at the bottom that magnifies the image that comes from the sample. The objectives can be of various magnifications. Example: 4.5X (magnifying glass), 10X, 40X and 100X (immersion objective).
The immersion objective is so named because it requires the placement of a few drops of oil between the objective and the sample. The others are called dry targets.
The objectives are printed with the characteristics they have.
Example: manufacturer's brand, field curvature correction, aberration correction, magnification, numerical aperture, special optical properties, immersion medium, tube length, focal length, coverslip thickness, and code ring Colour.
The lenses have a front lens located at the bottom and a rear lens located at the top.
Old microscopes are monocular, that is, they have only one eyepiece, and modern microscopes are binoculars, that is, they have two eyepieces..
The eyepieces are cylindrical and hollow in shape. These have converging lenses inside that expand the virtual image created by the lens..
The eyepiece joins the tube. The latter allows the image transmitted by the objective to reach the eyepiece, which will magnify it again.
The eyepiece in its upper part contains a lens called the eyepiece and in its lower part it houses a lens called the collector.
It also has a diaphragm and depending on where it is located it will have a name. Those that are located between both lenses is called Huygens eyepiece and if it is located after the 2 lenses it is called Ramsden eyepiece. Although there are many other.
The magnification of the eyepiece ranges from 5X, 10X, 15X or 20X, depending on the microscope.
It is through the eyepiece or eyepieces that the operator can view the sample. Some models have a ring on the left eyepiece that is movable and allows image adjustment. This adjustable ring is called a diopter ring.
It is the source of illumination and is located at the bottom of the microscope. The light is halogen and is emitted from the bottom up. In general, the lamp that microscopes have is 12 V.
The diaphragm of dark-field microscopes lack an iris; in this case, it prevents the rays coming from the lamp from reaching the sample directly, only the oblique beams will touch the specimen. Those beams that are dispersed by the structures present in the sample are those that will pass to the objective..
This explains why structures look bright and luminous in a dark field..
The condenser of a dark field microscope differs from that of a bright field.
There are two types: refractive capacitors and reflection capacitors. The latter in turn is divided into two categories: paraboloids and cardioids..
This type of condenser has a disc that is interposed to refract light rays, it can be located above the front lens or on the back side..
It is very easy to improvise a condenser of this type, since it is enough to place in front of the front lens of the condenser a disk made of black cardboard that is smaller than the lens (diaphragm)..
A brightfield light microscope can be converted to a darkfield microscope using this tip..
They are the ones used by stereomicroscopes. There are two types: paraboloids and cardioids..
-It is used to investigate the presence of Treponema pallidum in clinical samples.
-Also useful for observing Borrelias and Leptospiras.
-It is ideal for observing behavior in vivo of cells or microorganisms, as long as it is not necessary to detail specific structures.
-It is ideal for highlighting the capsule or wall of microorganisms.
-Refractive condenser darkfield microscopes are less expensive.
-Its use is very useful in 40X magnification.
-They are ideal for observing samples that have a refractive index similar to the medium in which they are found. For example, cells in culture, yeast or motile bacteria such as spirochetes (Borrelias, Leptospiras and Treponemas).
-You can see the cell in vivo, which allows to evaluate their behavior. For example, Brownian movement, movement by flagella, movement by emission of pseudopods, process of mitotic division, hatching of larvae, budding of yeasts, phagocytosis, among others..
-Allows to highlight the edges of structures, for example capsule and cell wall.
-Disaggregated particles can be analyzed.
-The use of colorants is not necessary.
-Special care must be taken when mounting the preparations, since if they are too thick, they will not be well observed.
-Image resolution is low.
-Darkfield microscopes using refractive condensers have a very low percentage of luminosity.
-To improve the image quality with an immersion objective (100X) it is necessary to reduce the numerical aperture of the objectives and thus increase that of the illuminating cone. For this, it is essential to incorporate an additional diaphragm that can regulate the numerical aperture of the objective..
-You cannot visualize dry slides, or colored slides, unless they are vital stains.
-It does not allow the visualization of certain structures, especially internal ones.
-Darkfield microscopes are more expensive.
Yet No Comments