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Section17:Microscopes
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Microscopes
A microscope is not designed magnify small objects. For example, you can find in any hobby or toy store a $49.95 instrument capable of magnifying objects to 1200 times. And that includes a zoom lens and light source. Most student & research microscopes magnify no more than 1000 times and cost starting at around $1500.00, with research microscopes going into the tens of thousands of dollars. Is the academic community being taken for a ride? No. The $49.95 microscope only gives you an image that is a soft blurs at 1000 times, whereas the research microscopes image is crystal sharp. This is called resolution, the ability to see fine details. Once you can resolve fine details then you can magnify them. Every optical system has a finite resolution, if you magnify objects beyond the resolution the result will be empty magnification. So, the actual purpose of a microscope is to see small things clearly.
A desirable attribute of a microscope is depth of field, which is the range of depth that a specimen is in acceptable focus. A microscope that has a thin depth of field will have to be continuously focused up and down to view a thick specimen. A third feature that a microscope has its mechanism for contrast formation. Contrast is the ratio between the dark and the light. Typically, most microscopes use absorption contrast, that is the specimen is subjected to stains in order to be seen. This is called bright field microscopy. There are other types of microscope that use more exotic means to generate contrast, such as phase contrast, dark field, differential interference contrast. The fourth desirable feature is a strong illumination source. The higher a microscope magnifies the more light will be required. Also, there will be more optical trade off leeway when there is more light present. The illumination source should also be at a wavelength (color) that will facilitate the interaction with the specimen. All microscopes fall into either of two categories based on how the specimen is illuminated. In the typical compound microscope the light passes through the specimen and is collected by the image forming optics. This is called diascopic illumination. Dissecting (stereo) microscopes generally use episcopic illumination for use with opaque specimen. The light is reflected onto the specimen and then into the objective lens. The four attributes of an optical system trade off with each other. Resolution and brightness is antagonistic towards contrast and depth of field. For example, you can not have maximum resolution and maximum contrast simultaneously. Theoretically speaking, if you had an infinite resolving system there would be no contrast to discern the image. It is up to the microscopist to decide which attribute is needed to view a particular specimen. All of which are controlled be the iris diaphragm.
Microscope Parts
Objectives
The objective lens is the lens that is closest to the object or specimen. It is essentially the information-gathering lens of an optical system. Therefore it is regard as the most important lens of the microscope. There are many different types of objective lenses. The most common and inexpensive is the achromat. This lens is usually found on student microscopes. It is corrected for spherical aberration for only green light. Chromatic aberration is corrected in only two colors. The apochromat objective is far superior and generally very expensive. Chromatic aberration is corrected for all three colors and it is spherically corrected for two colors. These objectives quite often will require a special compensating eyepiece. Semiapochromat objectives have correction in between the apochromat and achromat. Flat field or plano objectives compensate for curvature of field and are excellent for histology work. The flat field objectives can be optically constructed to be also an achromat, semiapochromat or apochromat. In the latter case the lens would be called a plano apochromat which is generally regarded as the finest lens available. The price of a single plano apochromat will run into the many thousands of dollars.
Each objective has information critical for the maximum resolution possible written on the side of the barrel. Generally the magnification is printed in the largest text with the manufacturer type designation. The second value is the numerical aperture. Beneath that, in a smaller font the tube length and the cover glass thickness is given. Any special information will also be added such as if it is an oil lens, infinity etc. The tube length usually 160 refers to the distance between the objective and the eyepiece in millimeters. It must be maintain if the aberrations are to be corrected. You can recognize a superior microscope if when adjusting the interpupillary distance you can see the eyepiece extend. This happens to maintain the proper tube length. The cover slip thickness usually around 0.17mm is also critical. This corresponds to a cover glass of No. 1.5. The more sophisticated objectives even have a cover glass compensation control that you dial in the thickness of the cover glass.
Condensers
The sub-stage condenser of a microscope is design to focus the light onto the specimen. In addition it must also fill the numerical aperture of the objective. Like objective lenses there are several different types. The most common being the Abbe condenser. This type is not corrected for optical aberrations. The achromatic condenser is corrected for both spherical and chromatic aberrations. Both types of condenser have their numerical aperture printed on the side. This needs to be of equal or greater value then that of the objective N.A., otherwise, the full resolution of the objective will not be utilized. Most substage condensers can use immersion oil like that of the objectives to achieve their full N. A. This is not recommended unless you are doing very demanding photomicroscopy work.
Iris Diaphram
The iris diaphragm is the most important single control on the microscope. There is a misconception that it is used to regulate the amount of light. The light intensity control is the sole means to adjust the brightness. The iris diaphragm is the resolution verses contrast control. It does this by varying the size of the numerical aperture of the objective lens. Usually, lenses such as those found on cameras have the iris diaphragm built in the objective lens. In a microscope objective the iris diaphragm would have to be very small, which would be difficult to manufacture. So the optical engineers put the iris diaphragm at the optical equivalent of being in the objective lens, in the condenser assembly. This is one of the reasons why the condenser lens has to be set at the correct distance to the objective. In addition the iris diaphragm controls the depth of field.
Eyepiece
The eyepiece is basically a projection lens system. There are three types generally used in light microscopy. The most common is the Huygenian type. This eyepiece is used with low and medium magnification and is designed to project the image into a human eye. Some of these eyepieces will have a long eyepoint, the spot where your eye should be, so you can focus with your glasses on. If you suffer from stigmatism you should ware your glasses while using the microscope. If you are near or far sighted then you can adjust the eyepiece for your personal correction using the diopter corrector and leave your glasses off. The second type of eyepiece is the compensating eyepiece and is generally used with apochromate or flat field objectives. These provide superior image quality. The third type is the photo eyepiece. These are designed to project a corrected image onto film plane in a camera. These are generally considered the finest of eyepieces. All eyepieces will have a relative magnification written on the side of the barrel. They range in magnification from 2.5X to 15X with the lower magnifications used with the photo eyepiece.
Field Limiting Aperture
The field limiting aperture is used to determine the correct position and center of the condenser lens. It is used in conjunction with the condenser centering knobs to place the illumination in the center. It also helps in reducing the amount of optical flare.
