1.4: Microscopy

Introduction

Microscopes are among the most useful tools used by biologists. In this lab, you will learn how the microscope works, its limitations, and practice using it. There are different types of microscopes that magnify from as low as 10x to as much as 650,000x!

You will work with 2 types of microscopes: a compound light microscope and a stereoscopic dissecting microscope.

Activity A: Handling and Care of the Compound Light Microscope

In the lab, the Compound Light Microscopes (LM) are in the glass cabinets along the wall of the lab room. It is best to always use the same microscope, so note the number on it. Take good care of your microscope by following these guidelines:

• When retrieving your microscope from the cabinet, pick it up using two hands. Put one hand under the base and one around the arm. It is heavy, and you always use two hands!
• Clean the lenses before each use with lens paper that is on the lab tables and at the front of the class.
• If a lens accidentally touches a slide or gets wet, immediately dry and clean the lens with lens paper.
• When you move the microscope on the lab table, lift it rather than trying to slide it.

When you are done using the microscope, follow these steps:

• Remove the slide from the microscope and put it back in the appropriate tray.
• Using the revolving nosepiece, turn it back to the scanning (4x) objective lens - red.
• Lower the stage all the way using the coarse focus adjustment knob.
• Turn off the light and then unplug the microscope.
• Wrap the cord and tuck it in so that it won’t come loose.
• Clean the lenses with lens paper.
• Using two hands, carry your microscope back and put it in the cabinet in the place where you picked it up. It will fit in sideways.

Activity B: The Compound Light Microscope

Figure 1: Oxnard College’s Accuvision 3000 - LED Compound Light Microscope Accu-Scope 3000-LED Instruction Manual (Public Domain)

The arm holds the magnifying portions on the top and the illumination and focusing portions on the bottom.

Lenses on top are called ocular lenses, and they magnify 10X. There are 2 of them, making this a binocular microscope.

Lenses below are objective lenses, and they are in a revolving nosepiece. When you rotate the nosepiece, each one will click into position. Look at them, and you will see the magnification of each written on the side - 4X,10X, 40x, 100X. Total magnification is the magnification of the ocular multiplied by the magnification of the objective. For example, with the 4X objective, total magnification would be 40X. Fill in Table 1 with the total magnifications for each lens and the name of each lens.

Field of view is the area of the slide that you can see. Higher magnification will give you less field of view, but you will see greater detail.

Light is directed through the condenser, the stage opening, the slide with the specimen, the objective lens, the ocular lens, and then to the retina of your eye.

Light can be controlled with the iris diaphragm adjustment control on the condenser, which opens and closes a hole, allowing more or less light through the condenser; this also affects contrast, and by the light adjustment knob on the left side of the base. Use medium to low amounts of light, since this affects color saturation, detail, and depth of field.

Place the slide that holds the specimen you are studying on the stage and hold it in position with the stage clips (not labeled in the diagram). You move the slide left/right with the x-axis stage adjustment knob and towards/away from you with the y-axis stage adjustment knob.

The Coarse Focus knob raises and lowers the stage to focus on the specimen. Always start with the 4x objective by raising the stage all the way with the Coarse Focus, but do not use the Coarse Focus with any of the other objective lenses. The Fine Focus Knob raises and lowers the stage very slightly with each turn. This is the knob that you use when looking through the microscope with the higher-powered objectives - 10x, 40x and 100x.

Activity C: Using a Prepared Slide - letter "e"

Follow these steps to get the letter “e” slide in focus with the scanning objective lens.

1. Get a slide from the front desk that has a letter “e” on it.
2. Put it on the stage and secure it with the stage clips.
3. Use the x and y-axis adjustment knobs to move the “e” so that it is centered over the hole in the middle of the stage.
4. Turn the nosepiece until the scanning objective lens (4x) colored red “clicks” into place.
5. Turn the coarse adjustment knob towards and away from you. This lowers and raises the stage. Raise the stage all the way while watching from the side as you do so.
6. Look through the ocular lenses, and you should see the “e” It will probably be out of focus and not centered.
7. Center it using the Stage Adjustment Knobs and focus using the Fine Focus only. Do not use the Coarse Focus for this.
8. Adjust the light using the Condenser and the Light Adjustment Knob to a level that is comfortable for your eyes, not too bright.

   

   Figure 2: Close-up of the slide positioned correctly under the microscope. (Photo by Joe Frantz)

9. Draw what you see in the circle.
   
   
10. Describe what it is like. Is it right-side up or upside down (inverted)? _________________________
11. Is it correctly oriented, or is it backward (reversed)? _________________________
12. When you use the Stage Adjustment Knobs to move it up, does it move up or appear to move down? ______________
13. When you use the Stage Adjustment Knobs to move it to the left, does it move the image to the left or does it appear to move to the right? ______________
14. Now turn the scanning objective (4x) to the low power objective (10x). Make sure it clicks into position.
15. Look through the ocular. The “e” may be slightly out of focus. Focus with Fine Focus only by moving it towards you and away from you.
16. Use the Stage Adjustment Knobs to center it.

17. Draw what you see in the circle.
    
    

18. If you wish, you can repeat these steps to look at it under the 40x (high power) objective.

Activity D: Plant Cells - Elodea and Preparing a Wet Mount

Elodea is a plant that lives in freshwater. Plants are multicellular autotrophs - meaning that they carry out photosynthesis to produce their own energy.

Plants have 3 structures that animal cells do not have: cell walls, chloroplasts, and a central vacuole.

1. Obtain a small leaf of Elodea.
2. Place it on a microscope slide.
3. Add a drop or 2 of water.
4. Carefully place a coverslip over the leaf.
5. Focus and center with the scanning (4x) objective lens.

   

   Figure 3: Making a wet mount (http://www.redwoods.edu)

6. Turn to the 10x, low power objective, focus and center.
7. Turn to the 40x, high power objective, focus and center
8. Draw and label - cell wall, cell membrane, chloroplasts, vacuole, cytoplasm, and, if visible, nucleus.

Activity E: Animal - cheek cells - squamous epithelium wet mount

Now you will make a wet mount of squamous cells that are found inside the mouth and in other locations. Animals are multicellular heterotrophs, meaning that they must consume energy from outside sources. They do not have chloroplasts, vacuoles, or cell walls. You will make a wet mount using the cells inside of your mouth (cheek)

1. Put 2 drops of water on a slide.
2. With a sterile cotton swab, carefully scrape a few cells from inside your mouth and smear into the water on the slide
3. Add a drop of methylene blue; it improves contrast and visibility.
4. Focus and center with the scanning (4x) objective lens.
5. Turn to the 10x, low power objective, focus and center.
6. Turn to the 40x, high power objective, focus and center.

7. Draw and label the cell membrane, cytoplasm, and nucleus.
   
   

Activity F: Depth of Field - crossed threads

Depth of field means the thickness of the specimen that appears in focus at one time. For example, if you are viewing an insect under the microscope, the top of the insect may be in focus while the legs underneath are blurry. As you adjust the fine focus knob, different layers come into focus.

Higher magnification will result in a shallower depth of field. Too much light will also have shallower depth of field.

So, when studying specimens to see all details, you will need to continuously change the focus using the fine focus knob.

Figure 4: Crossed threads. (http://www.redwoods.edu)

1. Obtain a slide labeled crossed threads.
2. Focus and center with the scanning (4x) objective lens.
3. Turn to the 10x, low power objective, focus and center.
4. Turn the fine focus toward you and away from you to ascertain the order of thread color from top to bottom.

In order from top to bottom, the colors are ______________, ______________, ______________

Activity G: Stereoscopic Dissecting Microscope

The stereoscopic dissecting microscope is used to study specimens that are too large and/or thick to be studied with a compound light microscope. They have a light source both above and below the specimen. They typically magnify to about 50x with a Zoom lens. Obtain an object such as a coin, rock, pencil, or even your finger and study it with the microscope. Draw what you see.

Label the parts of the stereoscopic dissecting microscope: Ocular Lenses, Focus Knob, Zoom Magnification Knob, and the Upper and Lower Light Switches.

Figure 5: The Stereoscopic Dissection Microscope. (Original Photo by Joe Frantz)

Additional Questions

Briefly describe how you will handle and care for your microscope each time you use it.

____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Give the function of each of these and when you would use them.

Condenser __________________________________________________________________________________________________________________________________________________________________________________________

Stage Adjustment Knobs __________________________________________________________________________________________________________________________________________________________________________________________

Coarse Focus __________________________________________________________________________________________________________________________________________________________________________________________

Fine Focus __________________________________________________________________________________________________________________________________________________________________________________________

Objective Lenses __________________________________________________________________________________________________________________________________________________________________________________________

Define these terms:

Depth of Field __________________________________________________________________________________________________________________________________________________________________________________________

Field of View __________________________________________________________________________________________________________________________________________________________________________________________

Wet mount __________________________________________________________________________________________________________________________________________________________________________________________

The Compound Light Microscope

Draw arrows to label the parts of the microscope. Label the Ocular Lenses, Revolving Nosepiece, Objective Lenses, Coarse Focus, Fine Focus, Condensor, Stage Adjustment Knobs.

Figure 6: The Compound Light Microscope. (Original Photo by Joe Frantz)

Table 1. Total magnification and Names of Objective Lenses

Name	Magnification	Ocular Magnification	Total Magnification
Scanning	4x	10x
Low Power	10x	10x
High Power	40x	10x
Oil Immersion	100x	10x

Licensing and Attributions

• CC Licensed Content, Original
• Biology Labs. Authored by: Joe Frantz. Provided by Oxnard College. Located at http://www.oxnardcollege.edu. License CC BY: Attribution
• Public Domain Content: Accu-Scope 3000-LED Series Instruction Manual: accu-scope.com Public Domain: No Known Copyright

This document, "Lab 4: Microscopy," serves as a comprehensive guide for students to understand and utilize various microscopes. It covers the fundamental principles of microscopy, practical handling and care instructions, and detailed activities for observing different types of cells and specimens.

Key Sections and Activities:

• Introduction: Provides an overview of microscopes, their magnification capabilities, and the types of microscopes to be used in the lab, specifically compound light microscopes and stereoscopic dissecting microscopes.
• Activity A: Handling and Care of the Compound Light Microscope: Outlines essential guidelines for retrieving, cleaning, moving, and storing the compound light microscope, emphasizing two-handed handling and proper lens cleaning techniques.
• Activity B: The Compound Light Microscope: Describes the various parts of a compound light microscope, including the arm, ocular lenses, objective lenses, revolving nosepiece, stage, condenser, and focus knobs. It also explains key concepts such as total magnification and field of view.
• Activity C: Using a Prepared Slide - letter “e”: Provides step-by-step instructions for focusing on a prepared "e" slide using different objective lenses and observing how the image is inverted and reversed. Activity D: Plant Cells - Elodea and Preparing a Wet Mount: Guides users through the process of preparing a wet mount of Elodea plant cells and identifying structures like cell walls, chloroplasts, and vacuoles.
• Activity E: Animal - cheek cells - squamous epithelium wet mount: Instructs on preparing a wet mount of human cheek cells and identifying the cell membrane, cytoplasm, and nucleus. 6 Page 10 Page
• Activity F: Depth of Field - crossed threads: Explains the concept of depth of field using a crossed threads slide and demonstrates how to determine the order of threads.
• Activity G: Stereoscopic Dissecting Microscope: Introduces the stereoscopic dissecting microscope for observing larger specimens and includes an activity for users to study an object and label the microscope's parts.

Additional Questions and Tables: Includes questions about microscope handling, the functions of various parts, definitions of key terms, and a table for calculating total magnification.