To magnify an object, light is projected through an opening in the stage, where it hits the object and then enters the objective. An image is created, and this image becomes an object for the ocular lens, which remagnifies the image. Thus, the total magnification possible with the microscope is the magnification achieved by the objective multiplied by the magnification achieved by the ocular lens.
Most microscopes are parfocal. This term means that the microscope remains in focus when one switches from one objective to the next objective.
The ability to see clearly two items as separate objects under the microscope is called the resolution of the microscope. The resolution is determined in part by the wavelength of the light used for observing. Visible light has a wavelength of about nm, while ultraviolet light has a wavelength of about nm or less.
The resolution of a microscope increases as the wavelength decreases, so ultraviolet light allows one to detect objects not seen with visible light.
The resolving power of a lens refers to the size of the smallest object that can be seen with that lens. The resolving power is based on the wavelength of the light used and the numerical aperture of the lens. The numerical aperture NA refers to the widest cone of light that can enter the lens; the NA is engraved on the side of the objective lens. If the user is to see objects clearly, sufficient light must enter the objective lens.
Therefore, the object is seen poorly and without resolution. With the increased amount of light entering the objective, the resolution of the object increases, and one can observe objects as small as bacteria.
Resolution is important in other types of microscopy as well. Other light microscopes. In addition to the familiar compound microscope, microbiologists use other types of microscopes for specific purposes. These microscopes permit viewing of objects not otherwise seen with the light microscope. This microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle.
A light object is seen on a dark background. The final image remains inverted, but it is farther from the observer, making it easy to view the eye is most relaxed when viewing distant objects and normally cannot focus closer than 25 cm. This equation can be generalized for any combination of thin lenses and mirrors that obey the thin lens equations. The overall magnification of a multiple-element system is the product of the individual magnifications of its elements.
Calculate the magnification of an object placed 6. The objective and eyepiece are separated by This situation is similar to that shown in Figure 2. To find the overall magnification, we must find the magnification of the objective, then the magnification of the eyepiece. This involves using the thin lens equation. Isolating d i , we have. Substituting known values gives. The object distance is the distance of the first image from the eyepiece.
This places the first image closer to the eyepiece than its focal length, so that the eyepiece will form a case 2 image as shown in the figure.
Both the objective and the eyepiece contribute to the overall magnification, which is large and negative, consistent with Figure 2, where the image is seen to be large and inverted. In this case, the image is virtual and inverted, which cannot happen for a single element case 2 and case 3 images for single elements are virtual and upright. The final image is mm 0. Had the eyepiece been placed farther from the objective, it could have formed a case 1 image to the right. Such an image could be projected on a screen, but it would be behind the head of the person in the figure and not appropriate for direct viewing.
The procedure used to solve this example is applicable in any multiple-element system. Each element is treated in turn, with each forming an image that becomes the object for the next element. The process is not more difficult than for single lenses or mirrors, only lengthier.
The lenses can be quite complicated and are composed of multiple elements to reduce aberrations. Microscope objective lenses are particularly important as they primarily gather light from the specimen. Three parameters describe microscope objectives: the numerical aperture NA , the magnification m , and the working distance. Figure 3. While the numerical aperture can be used to compare resolutions of various objectives, it does not indicate how far the lens could be from the specimen.
The higher the NA the closer the lens will be to the specimen and the more chances there are of breaking the cover slip and damaging both the specimen and the lens. The focal length of an objective lens is different than the working distance. This is because objective lenses are made of a combination of lenses and the focal length is measured from inside the barrel. The working distance is a parameter that microscopists can use more readily as it is measured from the outermost lens. The working distance decreases as the NA and magnification both increase.
In photography, an image of an object at infinity is formed at the focal point and the f -number is given by the ratio of the focal length f of the lens and the diameter D of the aperture controlling the light into the lens see Figure 3b. If the acceptance angle is small the NA of the lens can also be used as given below. As the f -number decreases, the camera is able to gather light from a larger angle, giving wide-angle photography.
As usual there is a trade-off. In optical fibers, light needs to be focused into the fiber. Figure 4 shows the angle used in calculating the NA of an optical fiber.
Figure 4. Light rays enter an optical fiber. Figure 5. Light rays from a specimen entering the objective. The water and oil immersions allow more rays to enter the objective, increasing the resolution. Can the NA be larger than 1.
This minimizes the mismatch in refractive indices as light rays go through different media, generally providing a greater light-gathering ability and an increase in resolution. Figure 5 shows light rays when using air and immersion lenses.
When using a microscope we do not see the entire extent of the sample. Depending on the eyepiece and objective lens we see a restricted region which we say is the field of view.
The objective is then manipulated in two-dimensions above the sample to view other regions of the sample. Electronic scanning of either the objective or the sample is used in scanning microscopy. These lenses have a magnification power of four, 10, 40 and , respectively. The shorter the lens, the lower magnification power it has.
These four lenses are interchangeable and usually parfocal, meaning you never lose focus of the image even while changing from one lens to the next. The ocular lens, or eyepiece lens, is the one that you look through at the top of the microscope. The purpose of the ocular lens is to provide a re-magnified image for you to see when light enters through the objective lens.
The ocular lens is generally or times magnification. The power of the ocular lens combines with the objective lens to allow a much larger and clearer image, with a total magnification assuming the ocular lens has times magnification of 40, , , and times.
The condenser lens focuses light from the light source onto the slide or object, which feeds into the objective lens. The condenser lens is under the slide platform and above the light source. The amount of light allowed into the condenser lens can be altered by using the diaphragm. The amount of light allowed in will need to be adjusted whenever you use a different objective lens to see the object.
Condenser lenses are more useful when the magnification is times or higher, and best when using an oil-immersion lens.
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