Marine Invertebrate Zoology Microscope Introduction

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Marine Invertebrate Zoology Microscope Introduction Introduction A laboratory tool that has become almost synonymous with biology is the microscope.

1 Marine Invertebrate Zoology Microscope Introduction Introduction A laboratory tool that has become almost synonymous with biology is the microscope. As an extension of your eyes, the microscope is one of the most important biological tools that you will use. Get to know it, how it works, and what its limitations are. A light microscope is really only a sophisticated arrangement of magnifying lenses, constructed for convenient observation of small objects and using light as a source of illumination. There are several common types of microscopes, differing primarily in their magnification ranges, types of light used, physical structures, and applicability to the materials under study. Whatever type of microscope you will be using, proper care and maintenance will enable you to see the objects of study more clearly. All the lenses of your microscope should be cleaned before every use. Use only lens paper specifically manufactured for this purpose, to avoid scratching the surface of the lens. The eyepiece may often become smeared with mascara, dust, oil, or dirt. The objective lenses should be cleaned to remove any residue that may have coated them during previous use. Should a lens accidentally contact the object you are observing, immediately dry and clean the lens with lens paper. If seawater has contaminated a lens or the stage of the microscope, clean and dry it using distilled water and lens paper. The Compound Microscope A compound microscope is a delicate and expensive instrument and must be treated gently. Remove the compound microscope from its cabinet by the arm. Holding it upright and supporting the base with your free hand, take the instrument to your desk and put it down gently. A microscope should always be carried in this fashion with both hands. Familiarize yourself with the parts and operation of your instrument, referring when necessary to the microscope schematic on the following page. Microscope Introduction 1

2 Microscope Introduction 2

Parts of the Microscope The arm supports the body of the microscope, which in turn supports the magnifying elements.

The other magnifying elements are screwed into a revolving nosepiece at the bottom of the body tube (see picture 8 below). These magnifying elements are called objectives.

Below the body tube and nosepiece is a flat plate, the stage, where the objects to be examined are placed (see picture 5 below).

Light plays an extremely important role in the operation of a compound microscope.

3 Parts of the Microscope The arm supports the body of the microscope, which in turn supports the magnifying elements. At the top of the body tube is the ocular, or eyepiece, one of the magnifying elements (see pictures 10 & 11 below). It is usually removable. The other magnifying elements are screwed into a revolving nosepiece at the bottom of the body tube (see picture 8 below). These magnifying elements are called objectives. Together, the ocular and the objectives constitute the magnifying system of your microscope. Below the body tube and nosepiece is a flat plate, the stage, where the objects to be examined are placed (see picture 5 below). Directly beneath the stage opening you will notice a system of lenses, the condenser lens that serves to concentrate light from the light source below. Light plays an extremely important role in the operation of a compound microscope. Light is directed up through the stage opening, passes through the specimen, and then into the body tube, ultimately forming an image on the retina, the light-sensitive portion of the eye. The critical importance of light necessitates careful adjustment, and several controls are available for this purpose. The iris diaphragm may be found attached below the condenser. Look up through the stage opening, find the iris diaphragm lever, a small handle at one side of the diaphragm (see picture 9 below). Push it back and forth, noting how the size of the iris opening changes to regulate the amount of light passing through it. This control is one of the most important on your instrument. Open the diaphragm fully and reposition your microscope. Microscope Introduction 3

Changes and adjustments in the focus, which are necessary when an object is first placed on the microscope, are essential in order to obtain a precise image.

4 Just as with a magnifying glass, viewing an object with the microscope requires that the lens be a certain specified distance from the object. That distance is a property of the lens system and is constant for each objective; it is called the working distance. At the working distance from an object, an objective is in focus. Changes and adjustments in the focus, which are necessary when an object is first placed on the microscope, are essential in order to obtain a precise image. Focusing is accomplished by means of coarse and fine adjustment knobs, usually located on the arm (see picture 12 below). Try turning each of these knobs, noting carefully how each affects the position of the objectives. The coarse adjustment is used to obtain an approximate focus and the fine adjustment to obtain an exact and clear focus. Objects can be moved while on the stage using the x and y adjustments. Remember, that a mirror is used to get the image from the objectives to the ocular, which means that the specimen will appear inverted. Additionally, an adjustment on the x plane to the north will result in the apparent movement of the specimen to the south. Magnification The magnification of most objectives and oculars is engraved on them. On the ocular, the marking can be found on the smooth cylinder that fits inside the body tube. On the objectives, the magnification is engraved on the side of the cylinder. The marking means that the lens yields an image larger in each dimension than the object being viewed. Microscope Introduction 4

5 Remember that with a microscope of this kind you are using two sets of magnifiers. On low power (10X), for example, the objective forms an image (inside the body tube) that is 10 times as large as the viewed object; the 10X ocular then magnifies this primary image another 10 times. The image that finally reaches your eye has been enlarged 100X the size of the object. The total magnification for any combination of objective and ocular can be computed simply by multiplying the magnification of each lens (see calculations on last page). When using the microscope we will need to measure the size of certain objects. Remember that 1mm = 1,000µm (micron), or 1/25,400 inch. Since our scopes do not have ocular micrometers we will need to measure our field of view for each objective and then estimate the size of a given specimen (see calculations on last page). Oil Immersion Modern microscopes commonly have three or four objective lenses. Most microscopic observations use objective lens magnifications up to 45x power. However, when observing bacteria, you must use objective lenses up to 100x power. Owing to the small lens diameter of this power, special means must be employed to ensure that light coming up through the stage is not scattered and lost. Directing light into the objective lens is accomplished by the technique of adding light-transmitting oil to the slide and lowering the objective lens until it comes in contact with the oil. This keeps light from scattering out of the pathway of the objective lens. As stronger objective lenses are used, the focusing distance between the lenses and the slide gets smaller and smaller (fig. B.2). We will not be using the oil immersion lens in this class. \ The Dissecting Microscope The dissecting microscope is useful for observing organisms larger than those for which the compound microscope is used. The magnification is not as great, but there is much more distance between the objective Microscope Introduction 5

6 lens and the stage, allowing for manipulation and dissection of specimens. Most dissecting microscopes are binocular, providing a stereoscopic view, and are equipped with either a zoom lens or multiple objective lenses for variable magnification. Total magnification is computed in the same manner as for the compound microscope. There is no fine adjustment knob on these instruments. Refer to Figure B3 on the following page for Dissecting Microscope parts. Scanning Electron (SEM) and Transmitting Electron Microscopes (TEM) Electron microscopes are used to overcome many of the limitations found when using standard light microscopy. Electron microscopes fire a finely concentrated beam of electrons at a specially prepared specimen greatly increasing the magnification and resolution of the resulting image (Magnification can reach over 500,000X). The use of these instruments requires specially prepared rooms (no overhead electrical wiring, concrete room foundations that are deeper and poured separate from the attached building) and expertise much greater than standard microscopy. For more information on electron microscopy, UNCW s microscope laboratory has both and SEM and TEM in use. Visit: for more information. Microscope Introduction 6

7 Compound Microscope Quick Reference Initial Set-up 1. Unwrap scope and clean lenses using lens paper only 2. Turn light source on to intermediate brightness 3. Open iris diaphragm 4. Move condenser to the base of the stage, then make a ¾ turn down 5. Set interpupillary distance Focus 1. Place specimen properly on the stage and secure using stage clip 2. Use BOTH of your eyes! 3. USE THE LOW POWER OBJECTIVE to center the image with the stage controls 4. Obtain focus using the coarse and then the fine focus adjustment 5. Switch to the next power, center the image and focus using FINE FOCUS ADJUSTMENT ONLY 6. Do not use the oil immersion lens (100X) Rules in using the microscope 1. Always carry with both hands 2. Clean lenses before and after each use 3. Never leave any water on the stage or scope 4. Always remove slides before storing the scope Other Adjustments Necessary for Optimum Resolution and Contrast. Adopted from Richard Fox Lander University Copyright 2001 To Customize the Diopter Setting 1. The focal lengths of the two eyepieces must be adjusted to conform to differences, if any, between your two eyes so that the images in both eyepieces are in focus simultaneously. Each eyepiece is equipped with a diopter adjustment ring by which it can be focused independently of the other eyepiece. 2. Adjust the interpupillary distance until it is correct for you. 3. With a test slide in position on the stage, rotate the 10X ocular lens into position. 4. Rotate the diopter adjustment ring (=tube length adjustment ring) of the left eyepiece until its numerical value is the same as your interpupillary distance. 5. Close your right eye and bring an object into clear sharp focus using the coarse and fine adjustment, as needed. 6. Close your left eye and bring the same object into focus using the diopter ring of the right eyepiece. 7. Record the two diopter numerical values so you do not have to repeat this process every time you use the microscope: Left = Right = Microscope Introduction 7

8 To Adjust the Height of the Condenser The condenser height should be set correctly to optimize light quality and resolution. The position of the condenser is often accidentally changed and you need to know how to return it to the proper position. Height is adjusted using the condenser adjustment knob (=substage focusing control). These are small black plastic knobs located on the both sides of the condenser mount, below the stage. Turn either knob to move the condenser up or down. 1 Place a slide on the stage of the microscope and position it for viewing. There must be a slide on the stage! 2. Turn on the substage lamp and increase the voltage until its indicator lamp glows green. 3. Rotate the turret so that the 10X lens clicks into position. 4. Focus on the specimen using the coarse and fine adjustment as needed. 5. Open the condenser iris diaphragm fully. 6. While looking through the eyepieces, rotate the field iris adjustment knob until you can see the outline of the field diaphragm in the eyepiece. It will be a 10-sided polygon. 7. Use the condenser control knob to raise or lower the condenser until the field diaphragm is in sharp focus. 8. This is the proper height for the condenser and it should not be moved from this setting. If it is inadvertently moved, go through the adjustment procedure again to restore its proper position. Microscope Introduction 8

9 Name Magnifications & Field of View Calculations Magnifications Ocular Red Objective (Low Power) Yellow Objective (Medium Power) Blue Objective (High Power) Total Magnification of ocular and objective Red Yellow Blue X X X X X X X 1. Measure the field of view with a ruler using the Red objective (Round to the nearest mm.). mm µm 2. Measure the field of view with a ruler using the Yellow objective (Round to the nearest mm.). mm µm 3. Calculate Blue field of view by using the ratio of yellow/blue magnification to yellow/blue diameter. Example: mm µm Low magnification = X Med. Magnification Low diameter in microns Organism Mag Diameter (µm) Foraminifera Diatom Radiolaria Sponge Spicule Dinoflagellate Times Across Size (µm) Microscope Introduction 9

Laboratory Introduction

Laboratory Introduction There are two basic categories of microscopes: light microscopes and electron microscopes. Light, or optical, microscopes require light waves to provide the illumination while electron