Week 1: Microscopy

Objectives

Be able to describe:

the proper techniques for use of the compound and dissecting microscopes

the proper use of the compound and dissecting microscopes

total magnification of a compound microscope

magnification, working distance, parfocal, parcentric, illumination, depth of field

how to prepare a wet mount

the differences between the compound and dissecting microscopes

Be able to identify:

the parts of the compound and dissecting microscopes and note their functions

Overview

The microscope remains a pivotal tool in learning and research in biology to this day. Typical prokaryotic or eukaryotic cells range from 0.001 mm to 0.05 mm in diameter, with only the very largest cells (0.1 mm) entering into the range discernible by the human eye. Thus, microscopes are needed for detecting the existence of most cells and for visualizing cellular components. You will be using the compound light microscope and the dissecting microscope (also called the stereomicroscope). Effective use of these microscopes is one of the fundamental skills required for studying biology (many of your TAs and professors still use these skills on a daily basis in their research, so don’t take them lightly!), and it takes some practice to master this skill. This laboratory will show you how to use these instruments to their best advantage. Complete the following tasks to understand how to properly use a compound microscope and a dissecting microscope. You will apply these basic skills in many of the subsequent weeks, so take full advantage of this first lab to become fully acquainted with these invaluable tools!

The compound microscope

Activity 1.1: Learning the parts of the compound microscope

Materials:

compound microscope

sample slide Procedure:

Microscopes are expensive and delicate scientific instruments, please use them with care and follow the proper handling procedure:

1. Ensure that your work area is clean and uncluttered. 2. Identify the parts of your compound microscope shown in Figure 1.1 and note the functions of

these parts (Table 1.1). 3. Carefully clean the glass slide, oculars, and objectives with lens paper only. Consult your TA if

there is a scratch or smudge on any of the lenses after you have cleaned them. You only need a small piece of lens paper to get the job done!

4. Place the sample slide on the stage, and use the metal slide holder to hold it in place. Centre the specimen under the objective.

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5. Adjust the distance between the oculars to match the distance between your pupils. The distance between your pupils is called the interpupillary distance. To take full of advantage of the two ocular lenses, you have to match the distance between your eyes with the distance between the two ocular lenses. Think of the oculars as a pair of binoculars: you can bring them closer or further apart, but in order to have them both working at once, you have to bring the separate visuals together. This may take some practice. Ask your TA for help if you are unsure if you are using the oculars properly.

6. ALWAYS start with the lowest power objective (the 4X objective lens) and the stage at its lowest position (adjust the position of the stage using the coarse adjustment knob).

7. Use the coarse adjustment knob to bring the specimen into focus. Use the coaxial stage control knobs to move the specimen. Take this time to practice using the various parts of the microscope, including the iris diaphragm and the condenser lens adjustment. What happens to the image when you use these?

8. Before switching to a higher objective lens, ensure that the desired part of the slide is in the centre of the field of view through the oculars and that it is in focus. Rotate the revolving nosepiece to the 10X objective lens.

9. Your microscopes are parcentric and parfocal, which means that if an object is centered and in sharp focus with one objective, it will be centered and in focus with another objective. When using higher-powered objective lenses (10X and 40X), use the fine adjustments knob to focus.

10. What happens to the diameter of your field of view when you switch from a lower to a higher objective?

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11. Why should you always start with the lowest objective lens in the viewing position and the stage at its lowest position before you start focusing on a specimen?

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12. Depending on the slide you grabbed, you were examining animal, plant, or fungus tissue under the

microscope as your sample slide. What features of the specimen on the slide do you think can be used to classify this as an animal, plant, or fungus? Comparing your thin section with those of neighbouring groups might help.

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Figure 1.1: Parts of the compound microscope

Table 1.1: Functions of the parts of the compound microscope

ocular (eyepiece) The uppermost lens or series of lenses through which a specimen is viewed. Most oculars have a magnification of 10x. A microscope with one ocular is called a monocular microscope, and a microscope with two oculars is called a binocular microscope. The distance between the oculars can be adjusted to match different distances between pupils. Many times, a trinocular head is added to a microscope for teaching or photography purposes. Some oculars contain a pointer or a micrometer disc that is used to determine the size of an object. Never touch the ocular lens with your fingers. Your microscope is binocular which means it has two eyepieces as opposed to a monocular scope which has only one eyepiece

nosepiece Revolves and holds the objectives. When changing objectives, always turn the nosepiece instead of using the objectives. The objectives will click into place when properly aligned

objectives Lower lenses attached to the nosepiece. The magnification of each objective is stamped on the housing of the objective. Never touch the objective lenses!

scanning objective Used for viewing larger specimens or searching for a specimen. Shortest objective, and usually magnifies an object 4x

low power objective Used for coarse and preliminary focusing. Magnifies an object approximately 10x

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high power objective

Used for final and fine focusing. Magnifies an object approximately 40x

diopter ring Used to adjust the focus in one eyepiece in order to compensate for differences in the level of focus of the viewer’s eyes.

magnification adjustment

Ring around the body of the dissecting microscope that adjusts the magnification.

stage Platform on which the slide is placed and secured with a slide holder. There is a hole in the center of the stage to allow light to pass through

coaxial stage controls

Allow the stage to move. Once the slide is in place, rotate the two controls to move the slide from side to side, or toward and away from yourself

light source (illuminator), light switch, intensity control

Light source serves as the source of illumination for the microscope. The switch turns the light source on and off, and there is a dial control for intensity of the light source next to the switch

iris diaphragm Regulates light entering the microscope, usually controlled by a mechanical lever or rotating disc

condenser A lens system found beneath the stage: used to focus the light on the specimen

coarse adjustment knob

Used to locate the specimen on scanning power only

fine adjustment knob

Used to focus the specimen at low and high power

draw tube Connects the ocular to the body tube

body / arm Holds the nosepiece at one end and includes the draw tube

coaxial stage controls

Allow the stage to move. Once the slide is in place, rotate the two controls to move the slide from side to side, or toward and away from yourself.

base The supportive portion of the microscope that rests on the laboratory table

Activity 1.2: Understanding magnification, working distance, and field diameter

Materials:

compound microscope

sample slide from previous activity Procedure:

1. The magnification of each lens is written on the side of the lenses. The ocular has a 10X magnification. The powers of the objectives are 4X, 10X, and 40X. The total magnification is calculated by multiplying the power of the ocular by the power of the objective. Calculate the total magnification for the various lenses below:

4X (scanning) objective: _______; field diameter = 4.5mm 10X (low power) objective: ______; field diameter = 1.8mm 40X (high power) objective: ______; field diameter = 0.45mm

2. The working distance is the distance between the objective lens and the specimen. As the working distance decreases, the magnification increases. More light is needed when the working

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area decreases. Describe what happens to the working distance and the brightness of the image of your sample slide when you change from the 10X to the 4X objective lens:

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3. The field diameter is the diameter of the circular field of view that you see when you look through

the oculars. This diameter changes at different magnifications. What happened to the diameter of the field of view when you changed from the 10X to the 4X objective lens?

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Activity 1.3: Orientation of specimens under the compound microscope

Materials:

compound microscope

prepared slides of the letter “e” Procedure:

1. Place the slide of the letter “e” on the stage. Sketch the letter “e” as you see it with the naked eye on the stage of your microscope. Observe the slide using the 4X objective lens. Beside the first sketch, draw the letter “e” as you see it in the oculars.

2. What is the difference between the two sketches?

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3. When you move the slide to the right while looking under the microscope, which way does the specimen on the stage move?

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4. Which way does the image move when you move the specimen towards you?

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Microscope lenses have a restrictive plane of focus, a specific distance from the lens where the specimen can be sharply focused. This plane of focus has some depth to it called the depth of field, so that only a part of a thick 3-dimensional object (even a specimen on a microscope slide has some 3-dimensionality to it) is in focus at any one setting. As a rule, the higher the magnification, the lower the depth of field.

Materials:

compound microscope

one slide with 3 different coloured threads Procedure:

1. Note the order in which the threads have been laid out on the slide, from first to last.

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2. Locate the point at which the three threads intersect on the slide and centre this position under the 4X objective lens.

3. Slowly focus until the first, then the second, and then the third thread comes into focus. In which order did the colours appear in focus?

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4. Switch to the 10X or 40X objective lens and notice that the depth of field becomes shallower. If you use the fine focus, the 3-dimensional form of the threads can still be determined.

The dissecting microscope

The dissecting microscope (Fig. 1.2) is also known as the stereomicroscope. The two separate lenses of the dissecting microscope allow one to see objects in three dimensions (i.e. stereoscopic vision). Dissecting microscopes do not magnify to the extent of compound microscopes. The two oculars usually are 10X in magnification and most student models have two objectives that provide 2X and 4X magnification, so the total magnification is 20X and 40X respectively, with either step or zoom magnification. This lower magnification also means that the depth of field is much greater for dissecting microscopes. Dissecting microscopes utilize two types of light: incident light (direct illumination) or transmitted light (from beneath the specimen). Opaque objects placed on the microscope stage can be directly illuminated with incident light from an illuminator. Your microscopes only come equipped with incident light. The large working distance between the objective lenses and the specimen being viewed allows for easier manipulation or dissection of the specimen. Another advantage of the dissecting microscope is that it gives a true image of the object that is neither reversed nor inverted.

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Figure 1.2: Parts of the dissecting microscope.

Activity 1.5: The dissecting microscope

Materials:

dissecting microscope

pinned insects (do not remove from box), Daphnia, isopods, pond water Procedure:

1. Identify the parts of the dissecting microscope using Figure 1.2. 2. Identify the functions of the parts of the dissecting microscope using Table 1.1. 3. Plug in the transformer to the electrical outlet. 4. Turn on the transformer and direct the light on to the specimen of your choice on the stage. Adjust

the light intensity on the transformer until you have the optimal image when using the oculars. 5. Adjust the magnification to its lowest power with the magnification adjustment on the top or side of

the microscope body. 6. Adjust the interpupillary distance of the ocular lenses. 7. Focus on the specimen(s) of your choice by lowering the microscope body (using the focusing

knob) to its lowest point and slowly raising the microscope body from this point until the image is the sharpest. Sketch your observations of the specimen(s) below.

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8. List three differences between the compound microscope and the dissecting microscope.

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Activity 1.6: Making a wet mount

Materials:

dissecting microscope

compound microscope

pond water and pipette

glass slide and cover slip

visual key to common microorganisms in pond water

Procedure:

1. Observe the sample of pond water under the dissection microscope on the side bench and look for microorganisms. Use the provided pipette and place a single drop of pond water containing microorganisms on to the middle of the slide (this exercise works best if you take pond water from the bottom of the jar where most of these organisms are situated).

2. Place the coverslip at a 45-degree angle, with one edge of it touching the water, and slowly lower the coverslip on top of the drop. This technique will help limit the number of bubbles that are stuck underneath the coverslip.

3. Identify any microorganisms you see using the dissecting microscope and compound microscope and sketch them below. Use the visual key provided on the lab bench to help you identify some of the more common ones.

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NOTE: At the end of each lab, ensure that you turn off the light source to any compound or dissecting microscopes you have used, remove any slides or specimens and place them back in the appropriate space provided, cover the microscopes, and place the 4X objective lens in the viewing position (for compound microscopes).