Part 1 - Get a Lab Appointment and Install Software:

Set up an Account on the Scheduler (FIRST TIME USING NANSLO):Find the email from your instructor with the URL (link) to sign up at the scheduler.Set up your scheduling system account and schedule your lab appointment. NOTE: You cannot make an appointment until two weeks prior to the start date of this lab assignment. You can get your username and password from your email to schedule within this time frame.

Install the Citrix software: – go to http://receiver.citrix.com and click download > accept > run > install (FIRST TIME USING NANSLO).You only have to do this ONCE. Do NOT open it after installing. It will work automatically when you go to your lab. (more info at http://www.wiche.edu/info/nanslo/creative_science/Installing_Citrix_Receiver_Program.pdf)

Scheduling Additional Lab Appointments: Get your scheduler account username and password from your email.Go to the URL (link) given to you by your instructor and set up your appointment.(more info at http://www.wiche.edu/nanslo/creative-science-solutions/students-scheduling-labs)

Changing Your Scheduled Lab Appointment: Get your scheduler account username and password from your email. Go to http://scheduler.nanslo.org and select the “I am a student” button. Log in to go to the student dashboard and modify your appointment time. (more info at http://www.wiche.edu/nanslo/creative-science-solutions/students-scheduling-labs)

Part 2 – Before Lab Day:

Read your lab experiment background and procedure below, pages 1-17.

Submit your completed Pre-Lab 1-3 Questions (pages 6-9) per your faculty’s instructions.

Watch the Microscope Control Panel Video Tutorial http://www.wiche.edu/nanslo/lab-tutorials#microscope

Part 3 – Lab Day

Log in to your lab session – 2 options:1)Retrieve your email from the scheduler with your appointment info or 2) Log in to the student dashboard and join your session by going to http://scheduler.nanslo.org

NOTE: You cannot log in to your session before the date and start time of your appointment. Use Internet Explorer or Firefox.

Click on the yellow button on the bottom of the screen and follow the instructions to talk to your lab partners and the lab tech.

Remote Lab ActivitySUBJECT SEMESTER: ____________

TITLE OF LAB: Introduction to Microscopy

Lab format: This lab is a remote lab activity.

Relationship to theory (if appropriate): In this lab you will learn the underlying principles behind the histological study of tissues.

Instructions for Instructors: This protocol is written under an open source CC BY license. You may use the procedure as is or modify as necessary for your class. Be sure to let your students know if they should complete optional exercises in this lab procedure as lab technicians will not know if you want your students to complete optional exercises.

Instructions for Students: Read the complete laboratory procedure before coming to lab. Under the experimental sections, complete all pre-lab materials before logging on to the remote lab. Complete data collection sections during your online period, and answer questions in analysis sections after your online period. Your instructor will let you know if you are required to complete any optional exercises in this lab.

Remote Resources: Primary – Microscope, Secondary – Introduction to Microscopy slide set.

CONTENTS FOR THIS NANSLO LAB ACTIVITY:

Learning Objectives......................................................................................................... 2Background Information ................................................................................................. 2-6Pre-lab Exercise 1: The Microscope ............................................................................... 6-7Pre-lab Exercise 2: Field of View and Depth of Field of a Compound Microscope ......... 7-8Pre-lab Exercise 3: Observation of Cells ........................................................................ 9Equipment ...................................................................................................................... 9Preparing for this NANSLO Lab Activity .......................................................................... 9-10Experimental Procedure ................................................................................................. 10Exercise 1: Operating a Compound Microscope ............................................................ 10-12Exercise 2: Field of View and Depth of Field of a Compound Microscope ..................... 13Exercise 3: Observation of Cells ..................................................................................... 14-15

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Summary Questions ........................................................................................................ 15-16Creative Commons Licensing .......................................................................................... 17U.S. Department of Labor Information ........................................................................... 17

LEARNING OBJECTIVES:

After completing this laboratory experiment, you should be able to do the following things:

1. Identify the parts of a compound microscope and operate it effectively.2. Understand the basic differences between Dissecting, Compound, and Electron

microscopes.3. Demonstrate the competency with the focus and operation of a microscope.4. Use a microscope to capture images and turn them into figures.

BACKGROUND INFORMATION:

There are few things that have impacted the development of modern biology more than the development of the microscope. Not only did the microscope open up a whole new world of observation which directly led to the discovery of microorganisms, cells, and some cellular components, but the microscope also affected the thought process and conduct of biological research. As Sachs wrote in History of Botany, “ . . . the use of the magnifying glass brought an advantage with it of a different kind - it taught those who use them to see scientifically and exactly.” (Julius Sachs, 1890) Additionally, three Nobel prizes have been awarded for the development of microscopes; the ultramicroscope won the award for chemistry in 1923, phase contrast microscopy won the award for physics in 1953, and electron and force microscopes won the award for physics in 1986 (Nobel Organization). Since the microscope is such an important instrument not only for scientific observation but for the development of the field of biology, we will spend a little time on the history and development of the instrument.

Humans have had at least a basic understanding of lenses for a long time. The existence of “burning glasses” and “magnifying globs” (hollow glass spheres filled with water) can be traced back almost 2000 years if not more (Singer, 1914). However, it was not until the 1300s, around the time we begin to see lenses used for eye glasses, that magnification started to be used by biologists (Singer, 1914). These earliest devices were single lens microscopes and had limited magnification compared to modern scopes. The pinnacle of this type of microscope was Leeuwenhoek’s discovery of “bacteria” in 1673 (Mazzarello, 1999).

During the 1600s, we begin to see the creation of the compound microscope which is a microscope with multiple lenses. The earliest of these microscopes were very similar if not identical to telescopes, being simple tubes with two or more lenses. The first practically used compound microscope was invented by the Janssens in 1590 (Singer, 1914). The compound

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microscope provided a much higher degree of magnification than the earliest scopes, however at a cost in clarity due to chromatic aberration and image distortion at higher magnification. This area of microscope development is most notably remembered for Hook’s detailed analysis published in his Micrographia in 1665 (Mazzarello, 1999), the work that coined the term “cells.”

The time period starting in the early 1800s to present has been predominantly focused on eliminating distortions and aberrations so that compound microscopes could be pushed to their absolute resolution limit (Singer, 1914). We will return to a discussion of the resolution limit of a compound microscope later. The first optical aberration we will discuss is chromatic aberration. This type of defect is caused because white light is actually composed of

many different colors of light. As an example, consider a prism. On one side white light goes in and on the other a rainbow comes out (Figure 1). This is because the degree of refraction (the bending of the light) is dependent on the wavelengths of the light. The shorter wavelengths are refracted less than the longer wavelengths. In this case, the prism is a basic lens. Therefore, in a compound microscope that uses white light, the different wavelengths that compose this light would focus at different points because they are each refracted a different amount. This produces a color blur around objects (chromatic aberration). Over many years this problem was fixed with the development of compound lenses which are composed of multiple lenses. In this case, each of the sub-lenses has a different refractive index. This brings the focal point of

the different wavelengths of light back together (Ernst, 1900). These are called achromatic or apochromatic lenses.

The other type of distortion is curvilinear distortion which is the tendency to bend straight lines into curves. An example that you are likely familiar with is the fisheye effects seen in wide angle photography. The reason for the distortion is that light bends as it

strikes a lens at a non-perpendicular angle. In order to get the high degree of magnification needed in early compound microscopes, the single lenses that were used had very curved surfaces. This meant that the light at the edge of the lens was bent much more than the light from the center of the lens. To correct this, microscope builders again used compound lenses.

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In this case, the sub-lenses have different shapes (Ernst, 1900). The different shapes will affect the bending of the light rays so that the light rays are brought back perpendicular to each other. Since the shape of a lens changes its effect on light, multiple lenses can be used to shape light in many different ways. Figure 2 shows the effect of different lenses on light rays.

Over the last several decades, the development of modern microscopes has begun to be focused on the resolution limit. The resolution of a microscope is defined as the ability of the observer (person or camera) to distinguish between two objects that are close together. In 1873, Ernst Abbe showed that the theoretical resolution limit of a diffraction microscope was half the wavelength of the light being used (Lipson, 2011). Therefore the resolution limit of an optical microscope is about 190 – 350 nm (the human eye responds to light with wavelengths of 390 – 700 nm). To get better resolution than is possible with a compound microscope, scientist started using electron beams with scanning and tunneling electron microscopes that have wavelengths 100,000 times smaller than visible light (Scherzer, 1949). Recently, work using lasers and quantum mechanics has begun to develop microscopes that “break” the resolution limit of optical microscopes; however, we will not discuss these types of microscopes further in this protocol.

Since the resolving power of a microscope also indicates how much magnification you theoretically have, the type of microscope you use depends on the type of observations you are conducting. We will now briefly cover the other types of microscopes you should be familiar with. In addition to the compound microscope, you should be familiar with the dissecting and electron microscopes.

Of the three types of microscopes, the dissecting microscope has the lowest magnification. The magnification range of a dissecting microscope is 10x to 40x. It provides a view of a fairly large sample for the purposes of detailed examination and/or dissection. Typical subjects might be small animals, whole flowers, or organ parts. Dissecting microscopes are used to magnify specimens of sizes 10 μm to 0.1 m. If a dissecting microscope has two ocular lenses on separate body tubes, it is a stereoscope dissecting microscope. The dissecting microscope uses visible light which is brought in from the side of the sample.

The compound light microscope has a magnification range that place it between the dissecting microscope and the electron microscopes. The magnification range is 40x to 2000x. Typical subjects might be cells, large cellular organelles, and bacteria. Compound microscopes are used to magnify specimens of sizes 200 nm to 5 mm. The compound microscope uses visible light which is brought in from below the sample. Since the light must shine through the specimens, it must be small or thinly sliced to obtain a good image. Since you will be using this type microscope exclusively in this lab, we will also discuss the components and parts of this microscope.

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In a modern compound microscope, there are three main optical components (Figure 3). They are the condenser (Figure 3a), objective (Figure 3b), and the eye piece (Figure 3c). The condenser focuses the light from the light source onto the underside of the sample. The objective contains a complex series of lenses that correct chromatic aberration and image distortion. The objective also gives part of the microscope’s total magnification. The objectives are mounted into a wheel that allows the user to select which objective they wish to use. The length of the objective is often related to its magnification with longer objectives having higher magnifications. One of the reasons that the objectives are different lengths is so that the microscope will be par-focal at all magnifications which means that the object that is in focus at 10X will also be in focus at 20X, 40X, 60X, etc. The eye piece gives the final part of

the magnification such that the total magnification of a microscope is determined by multiplying the objective by the eye piece. As an example, if you’re using a 10X eyepiece and 20X objective, your total magnification will be 10 x 20 = 200X.

The electron microscopes have the highest effective magnification of the microscopes we are discussing. Electron microscopes come in two types. The scanning electron microscope (SEM) and the transmission electron microscope (TEM) each type employs electron bombardment to image very small specimens. In the SEM, the electrons pass through a three dimensional specimen and are “read” using a detection device. A computer reconstructs the specimen image from the information gathered by the scanning process. The magnification range of an SEM can reach 200,000X and provide great detail. The TEM allows internal investigations of the internal structure of prepared specimens. The TEM can reach up to 50,000,000X which is a higher magnification range than the SEM. Electron microscopes are used to image samples that range from 1 nm to 100 μm in size.

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References:

Ernst, H.C. (1900). The Development of the Microscope, J. Boston Soc. Med. Sci. 4, 148–152.1.

Julius Sachs, I.B.B. (1890). History of Botany (1530-1860) (Clarendon press).

Lipson, A. (2011). Optical physics (Cambridge ; New York: Cambridge University Press).

Mazzarello, P. (1999). A unifying concept: the history of cell theory. Nat. Cell Biol. 1, E13–E15. Organization, N.P. Microscopes: Time Line.

Scherzer, O. (1949). The Theoretical Resolution Limit of the Electron Microscope. J. Appl. Phys. 20, 20–29.

Singer, C. (1914). Notes on the Early History of Microscopy. Proc. R. Soc. Med. 7, 247–279.

PRE-LAB EXERCISE 1: The Microscope

There are many types of microscopes available for biologist to use. The functionality of these microscopes is dependent on many factors. Which type of microscope you choose to use is dependent on the particular task you are trying to accomplish.

Pre-Lab 1 Questions:

1. One of the most common types of microscope in use today is the compound light microscope. What property of this microscope’s construction gives it the name “compound microscope”?

2. Complete the following table of magnifications.

Microscope Objective

Total Magnification10X Eye Piece 20X Eye Piece

4X 10X 20X 40X 60X

Use the microscope types in the following list to answer the following questions. The names may be used more than once.

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Dissection MicroscopeCompound MicroscopeScanning Electron microscopeTransmission Electron microscope

3. The _____________________ microscope has the highest magnification.4. The _____________________ microscope is used to examine a whole organ.5. The _____________________ microscope has two or more sets of lenses.6. The _____________________ microscope uses light from the side to illuminate its

sample.7. The _____________________ microscope can generate magnifications of 200,000X.8. The _____________________ microscope used to look at whole cells or larger cellular

organelles.9. Label the parts on the following diagram of a compound microscope.

10. You are trying to build a light microscope with the best resolution possible. To do this your light source is going to be a single wavelength of visible light. You have single wavelength light-emitting diodes in Infrared, Red, Orange, Green, Blue, and Violet. Which one will you use as your light source and why?

PRE-LAB EXERCISE 2: Field of View and Depth of Field of a Compound Microscope

It is important to remember that the sample you are looking at in your microscope is actual a three dimensions object it has height, width, and depth. In microscopy we refer to these dimensions as the X, Y, and Z axis. We need to remember this because the different objectives on a microscope will display different amounts of each of these axis. Therefore, when you are examining a sample under the microscope you need to know both how much of the sample is visible and what part of it you are viewing. We determine how much of the sample we are

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viewing based on the amount visible to the ocular or the camera, this area is called the field of view. The field of view is a plane parallel (the x and y axis) to the slide and can be easily quantified using a ruler. The ruler we use with a microscope is called a stage micrometer.

While it might seem intuitive the part of the sample you see is the part that is in focus, what we call the focal point. We always need to remember that there may be parts of the sample either above or below the focal point that we cannot see. The amount of the sample that is in focal point is called the depth of field (the Z axis). The depth of field is perpendicular to the field of view. However, depth of field is much harder to measure then field of view and we will only be dealing with depth of field qualitatively in this lab. Both field of view and depth of field are related to and affected by the magnification of the objectives. In this section you will explore the relationship between the objective’s magnification and the field of view and depth of field.

Pre-lab 2 Questions

1. How do you think the depth of field and field of view will change as magnification increases.

Scientists utilize a specific methodology when conducting experiments. This process is called the scientific method. Very simply, in the scientific method you ask a question, make a prediction, test that prediction, and then revise your prediction if necessary. Your answer to question one in this exercise is a prediction, and the remaining parts of this exercise allow you to test and revise your prediction. However, scientists craft their predictions in a specific form called a hypothesis. In order for a hypothesis to be a scientific hypothesis, it must be both logically valid and testable. One way to write a hypothesis is to use an If … Then … statement, an example of which would be If the field of view decreases in diameter Then the depth of field will increase in size. Using this information, complete the next pre-lab question.

2. Rewrite you your answer to the previous question into an If … Then … hypothesis.

To make measurements on the microscope, you first need to determine the actual size of the field of view. To do this, you will use a Stage Micrometer which is a microscope slide that has a small ruler drawn on it. You will use the stage micrometer to measure the field of view (the visible area) for each objective on the microscope. The micrometer you will be using is 1mm in total length with 100 divisions.

3. What is the length of one division on the stage micrometer? (Remember to list your units.)

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PRE-LAB EXERCISE 3: Observation of Cells

In the next exercise, we will look at actual biological samples. The slides we will use are: Elodea Leaf, Frog Skin, and Mixed Protozoa. We will use these samples to make observations using the microscope and examine the differences between different types of cells.

Pre-lab 3 Questions:

1. Do you expect to see any differences between the cells in the Elodea Leaf and the Frog Skin? What differences do you expect to see?

2. Rewrite your answer to question 1 in the format of an If … Than … hypothesis.3. Do you think the Protozoa will be more like plants or animals? Why?4. Rewrite your answer to question 3 in the format of an If … Than … hypothesis

EQUIPMENT:

Paper Pencil/pen Slides

o Letter "e", Whole Mounto Colored Threads, Whole Mounto Stage Micrometero Elodea Leaf, Whole Mounto Frog skin, Whole Mounto Mixed Protozoa, Whole Mount

Computer with Internet access for the remote laboratory and for data analysis

PREPARING FOR THIS NANSLO LAB ACTIVITY:

Read and understand the information below before you proceed with the lab!

Scheduling an Appointment Using the NANSLO Scheduling System

Your instructor has reserved a block of time through the NANSLO Scheduling System for you to complete this activity. For more information on how to set up a time to access this NANSLO lab activity, see www.wiche.edu/nanslo/scheduling-software.

Students Accessing a NANSLO Lab Activity for the First Time

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For those accessing a NANSLO laboratory for the first time, you may need to install software on your computer to access the NANSLO lab activity. Use this link for detailed instructions on steps to complete prior to accessing your assigned NANSLO lab activity – www.wiche.edu/nanslo/lab-tutorials.

Video Tutorial for RWSL: A short video demonstrating how to use the Remote Web-based Science Lab (RWSL) control panel for the air track can be viewed at http://www.wiche.edu/nanslo/lab-tutorials#microscope.

NOTE: Disregard the conference number in this video tutorial.

AS SOON AS YOU CONNECT TO THE RWSL CONTROL PANEL: Click on the yellow button at the bottom of the screen (you may need to scroll down to see it). Follow the directions on the pop up window to join the voice conference and talk to your group and the Lab Technician.

EXPERIMENTAL PROCEDURE:

Once you have logged on the microscope you will perform the following Laboratory procedures:

EXERCISE 1: Operating a Compound Microscope

In this exercise, you will learn how to operate a compound light microscope. When the exercise is over, you will understand how to position a sample, focus the microscope and change the magnification.

Data Collection:

1. Select the letter "e" whole mount slide (Slide Cassette 1: #2) from the microscope interface.

2. Place the letter e in the center of the field of view and focus on it with the 10X objective.

First, we are going to examine the effects of magnification.

3. In the space below is a diagram of the slide as it is currently loaded on the microscope (Figure 4 a). In box (Figure 4 b) draw the letter e as you see it in the microscope.

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4. What are the differences between the observed letter e and the letter e mounted on the microscope? (hint there are two differences)

5. Change to the 20X objective and capture an image of the letter e (If you can’t see any part of the e go ahead and move the slide). Paste that image below.

6. Change to the 60X objective and capture an image of the letter e (If you can’t see any part of the e go ahead and move the slide). Paste that image below.

Second, we are going to examine microscope movement.

7. Change back to 10X objective.8. Now using the microscope controls, click on the top button which moves the stage in

(towards the body of the microscope if you are observing through the PTZ camera) so that the e moves slightly. How does the e move with respect to the direction the stage moves?

9. This time, press the left stage control button. This will move the stage to the left (towards the slide loader if you are observing through the PTZ camera) so that the e moves slightly. How does the e move with respect to the direction the stage moves?

Analysis:

10. Describe the difference in your observations of the black lines that make up the letter e using the 20X and 60X objectives.

11. In the three diagrams below (Figure 5), assume that each dashed line represents the distance the stage will move a single click of the microscope control buttons. Using the sequence of buttons, draw where the e will be after the button clicks.

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EXERCISE 2: Field of View and Depth of Field of a Compound Microscope

Data Collection:

First, we are going to examine the depth of field.

1. Select the Colored Threads, Wholemount Slide (Slide Cassette 1: #1) from the slide loader tab.

2. Examine the slide with the 10X objective and describe what you see.3. How many of the threads can you get in focus using the 10X objective? Which of the

threads is on top, in the middle, and on the bottom? Capture an image, using your word processor, label the top, middle, and bottom thread. Paste it below.

Change to the 20X objective, how many of the threads can you get in focus simultaneously?

4. Capture an image and paste it below.

Second we will examine the field of view.

5. Select the Stage Micrometer slide from the slide loader tab, center, and focus the micrometer in the field of view.

6. Create a table to record the diameter of the field of view for each of the objectives.7. Select each objective in turn and measure the diameter across the middle of the visible

area. Record your measurements the table you created in step 9, each group member should collect a set of data. Paste your table with all the group's data below.

Analysis:

8. What happens to the depth of field as the magnification increases? 9. Take the table you created in step 10 and add two columns to it. In the first column,

average the diameter measurements for all your group's data. In the second column, convert the mm lengths into µm lengths.

10. What happens to the field of view as the magnification increases?11. Refer back to the If … Then … hypothesis you created in Pre-Lab Exercise 3, question 2.

Was your prediction correct? Explain why or why not12. Rewrite your hypothesis in light of the information you learned performing this

experiment.

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EXERCISE 3: Observation of Cells

Data Collection:

1. Select the Elodea Leaf, whole mount (Slide Cassette 1: #4) from the slide loader tab.2. Center and focus the sample in the field of view. Change to the 40X or 60X objective.

Capture an image of the leaf cells. Paste the image below.

In the previous exercise, we determined the diameter of your field of view. We will use this information to determine the size of the Elodea cells.

3. Create a table to record your data. 4. Change your objective to 40x and move to an area so that the leaf cells completely fill

the field of view. 5. Position the slide so that the left edge of a single cell is on the left edge of your field of

view. Count how many cells in a single row that reaches all the way across to the right edge of the field of view.

6. Repeat step 9 three more times with different rows of cells. Paste your table below.7. Select the Frog Skin, Whole Mount (Slide Cassette 2: #33) from the microscope

interface.8. Center and focus the sample in the field of view. Change to the 40X or 60X objectives.

Capture an image of the Frog Skin cells. Paste the image below.9. Select the Mixed Protozoa, Whole Mount slide (Slide Cassette 1: #6) from the

microscope interface.10. Find some protozoa on the slide. Center and focus the sample in the field of view.

Change to the 40X or 60X objective. Capture an image of the protozoa. Paste the image below.

Analysis:

11. Using your word processor and the image you captured in step 6, label all the structures you can identify in a leaf cell (examples: nucleus, cell membrane, cytoplasm, cell wall etc.). Paste the image below.

12. Next we are going to determine the size of the leaf cells. Start by averaging the number of cells it takes to fill your field of view from top to bottom. Insert that number below.

13. Next divide the diameter of your field of view by the average number of cells. This will give you the size of your cell. Insert that number below (make sure to include your units).

14. Using your word processor and the image you captured in step 12, label all the structures you can identify in a Frog Skin cell (examples: nucleus, cell membrane, cytoplasm, cell wall etc.). Paste the image below.

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15. Did your observations identify any differences between plant and animal cells? How do these differences compare to your hypothesis?

16. Using your word processor and the image you captured in step 14, label all the structures you can identify in a protozoa cell (examples, nucleus, cell membrane, cytoplasm, cell wall etc.). Paste the image below.

17. Based on your observation about the protozoa, are protozoa cells more like plants or animal cells? Explain.

Until fairly recently all organisms were classified into one of the five kingdoms of life (Monera, Fungi, Protista, Plantae, and Animalia). This classification system was based on physical appearance and in some cases physiology. More recent studies using DNA sequence comparison has shown that all living organisms belong to three Domains of life Bactria, Archaea, and Eukaryota. In this system, it was realized that Plants, animals, and Protista all belong to the Eukaryota domain.

18. In light of the idea that plants, animals, and Protista are all separate groups of the Eukaryota domain, does your observations about the Protista make more sense? Why?

SUMMARY QUESTIONS:

1. You are looking at a sample at 10X. When you increase the magnification to 40X, the sample is no longer in the field of view. Why might this happen, and how would you correct it?

2. If the compound microscope is par-focal, explain why sometimes when you go from the 10X or 20X object to the 60X object is the sample not in focus.

3. If an electron microscope has the highest resolution and magnification, why do we still use dissecting and compound microscopes?

4. Why does an electron microscope have a higher magnification and resolution than a compound light microscope?

5. Given the following slide, draw how the sample would appear while looking through the microscope (Figure 6).

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6. It is important to understand how light moves through a microscope. Complete the diagrams in Figure 7 to show the light path. In Figure 7a, draw the mirror that is needed to direct light to the ocular instead of the camera. In Figure 7b, draw the path that the light takes to reach the ocular. In Figure 7c, draw the path the light takes to reach the camera.

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For more information about NANSLO, visit www.wiche.edu/nanslo.

All material produced subject to:

Creative Commons Attribution 3.0 United States License 3

This product was funded by a grant awarded by the U.S. Department of Labor’s Employment and Training Administration. The product was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor. The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.

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