BIO203A_M1_L1_Microscopy-2

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National University College *

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203

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Biology

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May 8, 2024

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Lab 1: Microscopy Objectives  Demonstrate the correct use of a compound light microscope  Explain the concepts of magnification  Name the main parts of a compound light microscope  Observe the microscopic characteristics of the main groups of microorganisms Background Microscopy is a fundamental tool in the field of microbiology: as a science dedicated to those organisms invisible to the naked eye, microscopes are needed to observe and study them. The first microscope was invented in the early 17th century by Zacharias Janssen of the Netherlands. Anton van Leeuwenhoek, a Dutch clerk of a dry-goods store, was the first to use a single-lens microscope to observe microorganisms in a variety of specimens, including pond water and scrapings of his teeth. Today, microscopes are widely used in scientific research, medicine, and industry to study everything from cells to crystals. Parts of the Microscope The brightfield microscope you will use in this exercise is composed of several parts (Fig.1). Figure 1. Parts of the microscope BIO203A ed2717b529db1e22e1d0fe6925536cc6c38d74e2.docx Page 1 of 7

Its basic frame consists of a base (A) , a stage (B) to hold the slide, an arm (C) for carrying the microscope, and a body tube (D) for transmitting the magnified image. The illuminator (E) is the light source used to illuminate the specimen, and it is in the base. Above the light is the condenser (F) , which consists of several lenses that concentrate light on the slide by focusing it into a cone. The condenser has an iris diaphragm, which controls the angle and size of the cone of light. The objective lenses (G) are located on a rotating nosepiece and are used to magnify the specimen. The ocular or eyepiece lens (H) is located at the end of the tube that you look through to observe the specimen. Most modern microscopes are binocular, e.g. they have two ocular lenses. Handling of the Microscope When handling a microscope, it is important to follow certain rules to ensure proper use and to avoid damage to the instrument. Always be mindful of the microscope's delicate parts and handle it with care. Rules for the correct handling of the microscope include: 1. Always carry the microscope with two hands, one under the base and the other grasping the arm. 2. The microscope should be stored in the appropriate cabinet, with its arm pointing outside. 3. Always store the microscope with the stage as far from the lenses as possible, and the scanning (4×) lens down. 4. The lenses of the microscope can be cleaned only with lens paper. Any other paper, including Kleenex, may scratch the lens. 5. Be sure to clean any oil residue from the lenses and the stage before putting the microscope away. When retrieving a microscope, check if it was put away correctly and notify your instructor if it was not. 6. Do not twist the cable around the stage; just leave it to the side. Focusing the Microscope By moving the lens closer to the slide or the stage closer to the objective lens, using the coarse- or fine-adjustment knobs, one can focus the image. The larger knob, the coarse adjustment, is used for focusing with the low-power objectives (4× and 10×), and the smaller knob, the fine adjustment, is used for focusing with the high-power and oil immersion lenses. The coarse-adjustment knob moves the lenses or the stage longer distances. The area seen through a microscope is called the field of vision (FOV). The magnification of a microscope depends on the type of objective lens used with the ocular. Compound microscopes have three or four objective lenses mounted on a BIO203A ed2717b529db1e22e1d0fe6925536cc6c38d74e2.docx Page 2 of 7

nosepiece: scanning (4×), low-power (10×), high-dry (40× to 45×), and oil immersion (97× to 100×). The magnification provided by each lens is stamped on the barrel. The total magnification of the object is calculated by multiplying the magnification of the ocular (usually 10×) by the magnification of the objective lens. The most important lens in microbiology is the oil immersion lens. It has the highest magnification (97× to 100×) and must be used with immersion oil. Optical systems could be built to magnify much more than the 1000× magnification . As you use the microscope, you will see that as magnification increases, your field of vision (FOV) will become smaller. To calculate FOV, you need to know the field number of the lens. The field number (FN) in microscopy is defined as the diameter of the area in the intermediate image plane that can be observed through the eyepiece, and it is usually written on the objective lens together with the N.A. To calculate the FOV, simply divide the FN by the total magnification used. As you move from lower to higher magnification, you will notice that the sample will remain in focus or only minor adjustments need to be made. Parfocality refers to the property of a microscope lens where the focus remains unchanged as the magnification is changed. This makes the focusing process much easier. Resolution Resolution or resolving power refers to the ability of lenses to reveal fine detail or two points distinctly separated. An example of resolution involves a car approaching you at night. At first, only one light appears, but as the car nears, you can distinguish two headlights. The resolving power is a function of the wavelength of light used and a characteristic of the lens system called the numerical aperture. Resolving power is best when two objects are seen as distinct even though they are very close together. Resolving power is expressed in units of length—the smaller the distance, the better the resolving power. Resolving power= Wavelength of light used 2 × numerical aperture Smaller wavelengths of light improve resolving power. The effect of decreasing the wavelength can be seen in electron microscopes, which use electrons as a source of light. The electrons have an extremely short wavelength and result in excellent resolving power. A light microscope has a resolving power of about 200 nanometers (nm), whereas an electron microscope has a resolving power of less than 0.2 nm. The numerical aperture is engraved on the side of each objective lens (usually abbreviated N.A.). Increasing the numerical aperture (for example, from 0.65 to 1.25) improves the resolving power. The numerical aperture depends on the maximum angle of the light entering the objective lens BIO203A ed2717b529db1e22e1d0fe6925536cc6c38d74e2.docx Page 3 of 7

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