MICROSCOPY MICROSCOPE TERMINOLOGY
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1 1 MICROSCOPY Most of the microorganisms that we talk about in this class are too small to be seen with the naked eye. The instruments we will use to visualize these microbes are microscopes. The laboratory microscopes are binocular, compound, parfocal, light microscopes. Binocular microscopes have two eyepieces or oculars. Compound microscopes allow us to visualize small microbes through the use of a series of convex lenses (ocular and objective lenses). Parfocal microscopes allow specimens to remain in focus (mostly) as we change magnification. Light microscopes use visible light to visualize a specimen. MICROSCOPE TERMINOLOGY Magnification The magnifying power of a lens is defined as the ratio of the object s apparent size when viewed through the lens compared to how the object would appear to the naked eye at a distance of 25 cm. The magnifying power of a compound microscope is the product of the magnifying power of the ocular lens and the magnifying power of the objective lens used to view the specimen. For example, the magnification of a specimen viewed with the ocular (10X) and 40X objective lens is 400X. Resolution or resolving power This is the ability of the lenses to distinguish between objects that are close together. In other words, resolution allows us to distinguish fine detail and structure. The better a lens s resolving power, the closer together objects can be and still be distinguished as separate. The resolution of a lens is dependent on the wavelength of light (λ) passing through the specimen and the numerical aperture (NA) of the lens (ability to gather light). The numerical aperture of a lens is in part dependent on the medium the light passes through on its way to the lens. More about this later. Resolution = λ/(2 x NA) For wavelength (λ) we will use 500 nanometers. This represents the wavelength of green light from the visible light spectrum. For the oil immersion lens the NA is Resolution = 500nm/(2 x 1.25) = 200nm Decreasing λ or increasing the numerical aperture improves resolution by creating a smaller resolution number (meaning the distance between two points is lessened and the two points can still be observed as separate points). Since the average bacterial organism has a diameter of 1 to 2µm, we are therefore able to visualize these bacterial cells using our light microscopes, as they can resolve down to 0.2µm. Note: a bacterial cell is comparable in size to the mitochondrion we see in eukaryotic cells.
2 2 Contrast Contrast refers to the differences in color and light within an organism or between an organism and its environment. Because many organisms are colorless, stains can be used to increase contrast. Field of view (FOV) This is the area that can be viewed at one time through a given objective lens. FOV decreases as magnification increases. Depth of field This refers to the distance, up or down, on either side of the final (sharpest) plane of focus that the objective can be moved where the image still remains in acceptable focus. The greatest depth of field occurs when using the low power (10X). The depth of field decreases as the magnifying lenses increase. The depth of field is at its least when the oil immersion objective (100X) is in use. The depth of field for the oil immersion objective is less than 1µm. Focusing properly using oil immersion is therefore important. Working distance This is the space between the lowest point of the objective and the slide when in focus. Working distance is greatest when the low power objective is in use. It is appropriate to use the coarse adjustment knob under low power. Working distance decreases as magnifying lenses are increased. Do not focus with the course adjustment when using the high dry objective (40X) or the oil immersion objective. With these objectives in place, the working distances are small and course adjustment focusing could possibly drive the objectives into the slide. MICROSCOPE PARTS The oculars are also known as the eyepieces. These are what you look through in order to view a specimen. The ocular lenses magnify the specimen 10X. The arm of the microscope is to be used when transporting the microscope. The base of the microscope contains the light source. You can adjust the brightness of the light illuminating the specimen by using the rheostat. When carrying the microscope one hand is on the arm of the microscope and the other is supporting the base. The revolving nosepiece contains the objective lenses. The objectives on our microscopes are 4X, 10X, 40X (high dry) and 100X (oil immersion lens). The stage is the platform that the specimen sits on. Microscope slides are held in place on the stage by clips. The condenser focuses light onto the specimen. The condenser can be adjusted to control the amount of light passing through the specimen. The iris diaphragm lever can also be adjusted to control the amount of light passing through the specimen.
3 The coarse adjustment knob is used to focus the specimen on the stage. This knob works by lowering and raising the stage by relatively large increments. For this reason, the coarse focus knob can only be used with the two lower power objectives (4X and 10X). The fine adjustment knob changes the height of the stage by much smaller increments. It should be the only focus knob that you use when viewing specimens with the 40X and 100X objective lenses. MICROSCOPE GUIDELINES 1. Keep microscopes away from lit Bunsen burners. 2. You should support the arm and the base of the microscope when you are transferring equipment between lab bench and cabinet. 3. In order to focus on a specimen: Place specimen on the stage and secure it with the stage clips. You can use the stage controls to change the position of the specimen on the stage. Using the 4X objective lens focus on the specimen using the coarse focus knob and then fine focus if necessary. The light source should be set at a relatively low intensity. You will need to increase this as you go up in magnification. If a higher magnification is desired, move the revolving nosepiece such that the 10X objective lens faces the stage. Since these are parfocal microscopes, you should only need to make minor adjustments in focus. If a still higher magnification is desired, move the revolving nosepiece such that the 40X objective lens faces the stage. Now further adjustments to focus may only be made with the fine focus knob. When using the oil immersion objective, rotate the high dry objective half way between the high dry and the oil immersion objectives. Place a drop of immersion oil on the slide. The oil is used to help prevent the loss of light due to refraction of the light away from the objective. 4. Only use lens paper to clean microscope lenses. 5. Make sure that any immersion oil is cleaned off of the microscope prior to putting the microscope away at the end of class. Immersion oil should NEVER EVER touch the 4X, 10X, or 40X objective lenses. 6. Do not scoot your microscope on the lab bench. Instead, put your microscope on a piece of paper that will allow you to slide you microscope if necessary. 7. In order to prepare the microscope for returning to the cabinet: Lower the stage to the lowest position Make sure the stage clips are not hanging off the side of the stage Remove the slide from the stage. If it has oil on it clean it. Clean the lenses with lens paper; Clean the oil immersion lens until the last sheet of lens paper shows no remaining oil Return the scanning objective into place over the stage Turn off the power 3
4 4 Turn down the rheostat Wrap the cord neatly around the microscope as demonstrated by your instructor PROCEDURES 1. Using the focusing method outlined above, look at the silk thread slide with the scanning objective (4X). Focus as necessary. How many threads are present? Which one is on top? On bottom? Now look at this slide with the 10X and high dry objective lenses. Focus as necessary. Can you still see the same number of threads at one time? 2. For determining the size of microscopic specimens... To measure the diameter of the field of view (FOV) of the 4X objective lens, place a short metric ruler on the stage of the microscope and adjust until the ruler is in focus. The divisions (mm) of the ruler should be visible. Align one of these divisions with the edge of the field of view, and count the number of divisions between that edge and the opposite edge. Record the diameter of the field of view in mm. o Diameter of FOV (4X) objective: The diameter of the field of view (FOV) can then be calculated for the other objective lenses using the equation D1M1 = D2M2 where o D1 = diameter of FOV using the scanning lens (4X) o M1 = magnification of the scanning lens (4X) o D2 = diameter of FOV using another objective lens (i.e. 10X) o D1 = magnification of the other objective lens Using the above formula, calculate the diameter of the FOV for the other objective lenses: o Diameter of FOV (10X): o Diameter of FOV (40X): o Diameter of FOV (100X): 3. Once these calculations are done for your microscope, you can now estimate (roughly) the size of an organism. Focus on an organism using an objective that makes the organism large enough to view clearly. Make sure the organism completely fits within the FOV. Estimate the number of cells that would fit across the diameter of the FOV of your objective lens. Next divide the diameter of the FOV by the number of cells. For example, if 10 cells fit across the diameter of the FOV of the 10X objective, then the calculation would be: o Diameter of FOV = 1800 µm = 180 µm # of organisms 10 cells organism
5 5 Using the previous formula, calculate the size (length) of the following: o Euglena: o Paramecium caudatum: o Rhizopus nigrans zygospore: o Nostoc: From Professor Scott Rose and Microbiology (BIO 440) Laboratory Manual, Fall 2012, Sacramento City College.
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