2.2: Lab Exercise 2- The Microscope

Lab Summary: In this lab, you will learn how to use an essential tool in science—the compound light microscope. Your learning will include familiarizing yourself with the parts of the microscope and how to use them, how to mount a slide, proper and efficient technique for focusing a slide, and calculating field diameters, total magnification, and cell sizes.

Your objectives for this lab are:

• identify the parts of the microscope and list the function of each.
• demonstrate the proper techniques for use, care, and transportation of the microscope.
• define and demonstrate a working understanding of the concepts of total magnification, resolution,
• parfocal, field diameter, depth of field, and working distance.
• calculate total magnification and field diameter, measure field diameter, and estimate cell size.
• utilize microscopy skills to properly focus, view, and manipulate a variety of histology slides .
• explain why the microscope’s field of view decreases as you increase the magnification.

Background

Many organisms, such as bacteria, and parts of organisms (cells), that biologists study are too small to be seen with the human eye. We use microscopes to magnify specimens for our investigations. The word microscope means “to see small” and the first primitive microscope was created in 1595.

There are several types of microscopes, but most of our work will be done using a compound light microscope. This type of microscope uses visible light focused through two lenses, the ocular lens and the objective lens, to view a small specimen. Only cells that are thin enough for light to pass through will be visible with a light microscope in a two-dimensional image. Another microscope that you may use in lab is
a stereoscopic or a dissecting microscope. This type of microscope is used to view thicker and/or larger specimens, such as an insect, in 3D. Since you are viewing larger samples, the magnification range of the dissecting microscope is lower than the compound light microscope. Lastly, the microscopes we will use are considered “parfocal”, which means that the view tends to stay in relatively well focused as higher objective lenses with higher magnification are used.

Activity 2.1: Rules and Instructions for Using the Compound Light Microscope

Procedure for Activity 2.1: To keep our microscopes in proper working order, please follow the steps outlined below for their use, transport, and storage. Read the rules and instructions below to make sure you understand them fully before you proceed to the next activity.

Important general rules:

• Always carry the microscope with 2 hands—place one hand on the microscope arm and the other hand under the microscope base.
• Do not touch the objective lenses (i.e. the tips of the objectives as seen in Figures 1.1 and 1.2).
• Keep the objectives in the scan position and keep the stage low when adding or removing slides.
• It’s advisable to look at the microscope from the side when you are making large changes in the height of the stage.
• The objective lenses are to be cleaned only with special lens paper and lens-cleaning fluid.
• Do NOT play handyperson—no microscope is to be disassembled. Report malfunctions to your instructor.

To obtain a microscope from the laboratory cabinet:

• First clear an area on your lab bench for the microscope—avoid a crowded working area.
• The microscopes are numbered on the arm and should be returned to their numbered area in the cabinets.
• Carry the microscope with TWO hands: one hand on the arm and one hand on the base.
• Secure the electrical cord—do not let it hang off the table!

When returning the microscope to the laboratory cabinet:

• Lower the stage.
• Rotate the scanning objective (the 4X objective) into position over the stage.
• Remove your slide from the stage.
• Clean the slide and objective using the special lens-cleaning fluid and paper provided.
• Center the stage so that it does not project too far to either side.
• Secure the cord by wrapping it around the base/arm or by wrapping around the correct area on the back of the microscope.
• Replace the dust cover.
• Carry the microscope with TWO hands: one hand on the arm and one hand on the base.
• Return the microscope to the same cabinet area from which you obtained it making sure to put it in arm outward.

Activity 2.2: Explore Parts of the Microscope

Procedure for Activity 2.2: Understanding the parts of the microscope and how they work to help you view a specimen or slide is essential knowledge, which will help you utilize the microscope most efficiently.

1. Read the following descriptions and use Figure 2.1 to learn about the parts of the microscope and the functions of these parts.
2. Locate each of the parts on your microscope (i.e. the microscope you’re using today)
3. Practice locating parts of the microscope with these two quick online games:

https://www.purposegames.com/game/part-of-the-microscope-1-right-side and https://www.purposegames.com/game/part-of-the-microscope-2-left-side

Arm and Base: structural pieces of the microscope. When carrying a microscope always grasp it firmly with two hands; one hand should be under the base, and the other hand should be around the arm or holding the carrying handle on the arm. When moving a microscope on your lab bench, you should also lift and move. Do not slide your microscope across a table or lab bench as the vibration can misalign lenses and other delicate parts.

Ocular lens: the eyepiece through which you look through at the top of the microscope. Typically, standard eyepieces have a magnifying power of 10x. Optional eyepieces of varying powers are available, typically from 5x—30x. The eyepiece tube holds the ocular lenses in place above the objective lens. Binocular microscope heads typically incorporate a diopter adjustment ring that allows for the possible inconsistencies of our eyesight in one or both eyes. Binocular microscopes also swivel (Interpupillary Adjustment) to allow for different distances between the eyes of different individuals.

Objective Lenses: the primary optical lenses on a microscope. They range from 4x—100x and typically, include, three, four or five on lens on most microscopes. Objectives can be forward-facing or rear-facing.

Nosepiece: structure that houses the objectives. The objectives are exposed and are mounted on a rotating turret so that different objectives can be conveniently selected. Standard objectives include 4x, 10x, 40x, and 100x (also known as the oil immersion lens) although different power objectives are available.

Coarse and Fine Focus knobs: used to focus the microscope on the specimen being viewed. Increasingly, they are coaxial knobs—that is to say, they are built on the same axis with the fine focus knob on the outside. Coaxial focus knobs are more convenient since the viewer does not have to move the hand around to find a different knob.

*The coarse objective knob should only be used with the 4x and 10x objective lenses. *The fine objective knob may be used with all objective lenses.

Stage: the flat platform where the specimen or slide is placed. A mechanical stage is used when working at higher magnifications where delicate movements of the specimen slide are required. Do not push on the mechanical stage with your fingers, use the stage adjustment knobs described below.

Stage Adjustment Knobs: allow you to move the slide slowly in multiple directions. These knobs allow you to move the slide incrementally left and right, as well as toward the front and back, without touching the slide or stage directly.

Illuminator/ Light Source: the light source for a microscope, which is typically located in the base of the microscope. Most light microscopes use low voltage, halogen or LED bulbs with continuous variable lighting control located within the base.

Condenser: used to collect and focus the light from the illuminator on to the specimen. It is located under the stage often in conjunction with an iris diaphragm.

Iris Diaphragm: controls the amount of light reaching the specimen. It is located above the condenser and below the stage. Most high-quality microscopes include an Abbe condenser with an iris diaphragm. Combined, they control both the focus and quantity of light applied to the specimen.

Condenser Adjustment Knob: moves the condenser up or down to control the lighting focus on the specimen. DO NOT TOUCH THE CONDENSER FOCUS KNOB, unless directed to do so by your instructor.

Figure \(\PageIndex{1}\): Parts of the Compound Light Microscope (right side)

Figure \(\PageIndex{2}\): Parts of the Compound Light Microscope (left side)

Activity 2.3: Calculating Total Magnification

Magnification is the ratio of the image size with the microscope to the actual size of the object. When you say that the total magnification is 10x (or, you say or read TM10x), the image you see using the microscope is ten times bigger than viewing the specimen with the naked eye. Remember with a compound light microscope you are magnifying with two lenses, so to calculate the total magnification you multiply the ocular lens magnification by the objective lens magnification. 

Procedure for Activity 2.3: Use the information above and on your microscope to fill in Table \(\PageIndex{1}\). 

Table \(\PageIndex{1}\): Total Magnification

	Ocular Lens Magnification	Objective Lens Magnification	Total Magnification for this Objective Lens
Scanning lens (red)
Low-power lens (yellow)
High-power lens (blue)
Oil-immersion lens (white)

Procedure for Activity 2.4: Remember as you increase magnification, you will probably need to increase the illumination of the slide.

1. Rotate the objective lenses so that the scanning (4x) objective is pointing down at the stage.
2. Adjust the stage so that the aperture (the opening in the middle of the stage) is centered.
3. Place a letter “e” slide on the stage, using the stage clamps to secure it.
4. Center the specimen on your slide under the objective by moving the stage. Depending on the type of microscope you are using, there will be stage adjustment knobs below the back edge of the stage. To prevent stripping the gears, do not use your hands to move the stage directly.
5. Look through the ocular lenses and prepare to focus your slide:

A. Starting with the scanning objective (4x) positioned over the specimen, use the coarse adjustment knob to move the stage up until your specimen comes into focus.

1. Sharpen the focus with the coarse and/or fine focus knob.
2. Use the stage adjustment knobs to move the slide until it is in the desired location.
3. Measure the working distance in millimeters for this objective (write it in the space below) and draw what you see in the field of view.
4. Work with a partner for steps d and e. Have you partner watch to make sure you move your slide to the right using the mechanical stage adjustment knob. You will observe the slide while looking through the oculars. Which way does the “e” move? _______________________________________
5. Have you partner watch to make sure you move your slide away from you using the mechanical stage adjustment knob. You will observe the slide while looking through the oculars. Which way does the “e” move? ________________________________________________________________
6. Explain the inversion phenomenon that you have observed throughout Step A.

*Do NOT lower the stage before moving to the next objective lens.*

B. Increase the magnification by slowly rotating the low power (10X) objective into position over the specimen.

1. Sharpen the focus with the coarse and/or fine focus knob. We use parfocal microscopes, so only a minimal amount of adjustment is usually necessary.
2. Use the stage adjustment knobs to move the slide until it is in the desired location.
3. Measure the working distance in millimeters for this objective (write it in the space below) and draw what you see in the field of view.

*Do NOT lower the stage before moving to the next objective lens.*

C. Increase the magnification by slowly rotating the high power (40X) objective into position over the specimen.

1. Sharpen the focus, if necessary, with the FINE FOCUS KNOB ONLY. Again, only a minimal amount of adjustment is usually necessary.
2. Re-center the specimen in the field, if necessary.
3. Measure the working distance in millimeters for this objective (write it in the space below) and draw what you see in the field of view.
4. Before you remove a slide, always return the objective lens to the scanning lens. Move the objectives backwards from higher to lower power to avoid damaging the objective lenses and the slides.
5. Go back and look at the working distances you measured. What happens to working distance as you increase magnification?

D. If required, your instructor will provide directions for using the 100x/oil immersion lens.

Activity 2.5: Measuring and Calculating Field of View

The field of view is the amount of the specimen you see when you look through the objectives. The field of view decreases at higher magnifications. Field diameter is the distance across the entire lighted field (not just of the slide or image you are viewing).

Procedure for Activity 2.5

1. Place a plastic or paper ruler across the stage aperture so that the edge of the ruler is visible as a series of horizontal lines across the field diameter. Alternately, you may use a micrometer slide or counting slide. If this is the case, please ask your instructor for directions.

2. Measure the field size in millimeters for the 4X and 10X objective lenses.

3. Calculate the field size in micrometers (μm). 1 mm = 1000 μm

   • Scanning lens (4X) __________mm, ___________ μm 

   • Low power lens (10X) __________mm, ___________ μm

4. If you know the field diameter at one magnification, you can use that information to help you calculate the field diameter of another magnification. Now, use the formula below to calculate the field diameter for the high-power lens (40X) and the oil immersion lens (100X). Use your information for the 4X or the 10X objective lens as your known values (the A-values)

Formula: (Field Diameter A) x (Total Magnification A) = (Field Diameter B) x (Total Magnification B)

• High power lens (40X) __________mm, ___________ μm
• Oil immersion lens (100X) __________mm, ___________ μm

Activity 2.6: Estimating the Size of a Specimen

The size of a specimen can be estimated if you know how many specimens (such as cheek cells below) appear to fit across the field of view and the measurement of the field of view. Cell morphology (size and shape) can be used in the clinical setting to determine if abnormalities are present and to discern different types of cells from each other.

Procedure for Activity 2.6

1. Use your field diameters from Activity 2.5 to fill in the field diameters for the views shown below. Remember that total magnification = (ocular lens magnification) x (objective lens magnification)
2. Look below at Cell A and estimate how many cells will fit across the entire field. Make note of this number under the black circle. Repeat this step for Cell B and Cell C.
3. Use the formula below to estimate the size of Cell A, Cell B, and Cell C. Write your answers in the spaces provided. Estimation of cell size is performed using the following formula:
   • Estimated cell size = (field diameter) / (# of cell that fit across the entire field)
4. Do all estimates match exactly? If not, explain why not.

Activity 2.7: Exploring Depth of Focus

The depth of focus is the thickness of the specimen that remains in focus at a given magnification. Depth of focus decreases at higher magnifications.

Procedure for Activity 2.7:

1. Obtain a slide with three colored threads.
2. View it using the scanning or low power objective.
3. Determine which color thread is on the top, in the middle, and on the bottom. Hint: using the instructions for focusing, focus on the area where the three threads cross. Use the fine focus to discern the order of the threads.
   • On the top: _________________________________
   • In the middle: _______________________________
   • On the bottom: ______________________________
4. When looking at a specimen on a slide are you looking at a 2D or 3D view? _______________________

Activity 2.8: Making a Wet Mount

Throughout the semester, you will be expected to successfully make a number of simple slide preparations called “wet mounts.” The specimen to be observed is placed on a clean slide, a drop or two of water added, and a cover slip carefully placed over the water and specimen. Your instructor will demonstrate this technique.

Make a wet mount of your cheek cells or pond water following the procedure below:

For Pond Water:

1. Place a drop of pond water on a clean glass slide using a plastic disposable pipette. Add one small drop of cellulose, Proto-slow, or other protist-slowing agent, if available, and mix gently with a toothpick.
2. Cover the pond water area with a coverslip by gently placing the cover slide on at a 90o angle to limit air bubbles.
3. Wipe off any excess liquid on your slide.
4. Place the slide on the microscope to observe the specimen.
5. Observe your slide first with the scanning objective lens. Then, carefully switch to the low-power and high-power objectives as necessary.
6. Hopefully, you will see some live organisms in your pond water. If you view green material, it is likely plant material. Look to see if there is anything moving. In the space below, describe what you observed under the microscope and draw a simple picture.

For Cheek Cells:

1. Gently scrape the inside of your cheek with the edge of the toothpick.
2. On the middle of a glass slide, swab your toothpick over a square area (about 2.5 cm wide) to distribute your cheek cells.
3. Add a small drop of methylene blue to the area containing the cheek cells. Be careful not to touch the tip of the dropper (containing methylene blue) to the cheek cells to avoid contamination.
4. Cover the cheek cells area with a coverslip by gently placing the cover slide on at a 90o angle to limit air bubbles.
5. Wipe off any excess liquid on your slide.
6. Place the slide on the microscope to observe the specimen.
7. Observe your slide first with the scanning objective lens. Then, carefully switch to the low-power and high-power objectives, as necessary.
8. Your cheek cells should look like pancake shapes with uneven edges. In the space below, describe what you observe under the microscope and draw a simple picture of three cheek cells, including labels for the cell membrane, nucleus, cytoplasm, and nucleolus.