THEMICROSCOPE

Fundamentals of Light Microscopy

OBJECTIVES:

Upon completion of this exercise the student should be able to:

1. name the parts of a compound light microscope and describe their function

2

. know how to properly:

a. carry a microscope, clean the lenses, and prepare the microscope for use and storage;

b. set and control illumination;

c. focus on a specimen;

d. determine the total magnification, the size of the field of view at any magnification, and estimate the size of a specimen;

e. make reasonably accurate drawings of specimens observed through the microscope.

INTRODUCTION TO THE MICROSCOPE

IMPORTANT TERMS

 

compound microscope – magnifies objects using a series of lenses 

resolution – detail; clarity of an image; the ability to recognize two separate points as in fact being separate 

magnification – increase in the size of an object’s image 

depth of field – amount of thickness of the specimen that is in focus at any one time 

field of view – total area visible through the microscope 

permanent slide – prepared so that the object and coverslip are permanently attached to the slide (object is not alive) 

wet mount – freshly prepared slide; living object is dropped on slide in water/media and covered with a coverslip 

contrast – use of a dye or adjusting the amount of light to distinguish the specimen from the background 

Front of microscope

Rear of microscope

Functions of the parts of a compound microscope

The stage of the microscope, is a rectangular plate on which the slides are placed. There will be a hole in the stage through which the light will pass.

The stage finger assembly, are two metal strips laying flat on the upper surface of the stage, which will be used to hold slides. You should never place your slide under these strips, they are spring loaded to pinch/hold your slide in place gently so that the mechanical stage knobs can easily move the slide around the stage.

The mechanical stage adjustment knobs, these knobs are used to move the slide left and right or up and down on the stage to allow you to view the whole slide. Generally, the knobs are located below the stage, on the opposite side of the microscope from the condenser adjustment knob.

The condenser is just under the stage. It will gather the light from the illuminator and focus it on the specimen laying on the stage. The condenser is mounted on a track and can be moved up and down beneath the stage.

The condenser adjustment knob controls the height of the condenser and is located just beneath the stage on the side opposite the mechanical stage adjustment knobs.

The iris diaphragm control ring or lever, is a rotating device attached to the condenser, or a metal tab or arm sticking out horizontally from the condenser. The iris diaphragm allows the amount of light passing through the condenser to be regulated. This device is very important, and you will need to adjust it most every time you change slides and change magnifications. The amount of light passing through the iris diaphragm is critical for proper viewing of the image.

The objective lenses are on a turret or nosepiece just above the stage. They magnify the image of the specimen to varying degrees. Each lens will have thin stripes of a different color. They will also have their magnification level etched on them (4x, 10x, etc.). The objective lenses ranging from 4x to 40x are “dry” lenses. The objective lens of 100x is an “

oil immersion

” lens, and before it can be used, a special oil must be added to the slide.

The nosepiece holds 3 to 4 objective lens and allows these lenses to be rotated into use. Usually you will feel the lenses “click” into place when they are properly aligned.

The ocular lens in the eyepiece. It further magnifies the image that has already been magnified by the objective lenses. It commonly magnifies 10x. It should contain a pointer (often in only 1 of the 2 lenses), which appears as a dark line when viewed through the microscope. The pointer can be used to specifically indicate features within the specimen.

The coarse focusing adjustment knobs and the fine focusing adjustment knobs on either side of the arm just above the base. There is one of each on each side. The coarse adjustment knobs are usually the larger of the two. These knobs are used to focus the specimen. Note that it doesn’t matter whether you use the right- or left-hand coarse adjustment knob. They are interlocked, thus they move in unison. When you turn the coarse focus knobs the stage is moved rapidly with poor precision. This focus knob is only used with the lowest power objective lens.

The fine focusing adjustment knobs move the stage is moved very slowly with great precision and are used to bring your specimen into precise focus. Usually they are located as the inner knob with the coarse focusing adjustment knob. Use this knob with the higher magnification objective lenses.

The arm is the main part of the microscope that holds the ocular lenses above the stage. Some microscopes have a handle in the back part of the arm. To carry a microscope properly you need to hold the arm directly (or the handle if available) and the base of the microscope.
NEVER hold the microscope by the stage when moving a microscope.

The base of the microscope is the bottom part containing the plug and the light source.

The light source is located in the base of the microscope and shines the light up through the condenser, iris diaphragm to the stage. It is controlled by both an on/off switch and a variable, rheostat knob.

Rheostat control knob (dimmer switch) is located on the base of the microscope and control the amount of light that reaches the condenser. At low power you will need to turn this down to decrease the amount of light on your slide but as you move to higher magnification objective lenses you will need to increase the amount of light.

CARE OF THE MICROSCOPE 

Microscopes are very expensive and delicate pieces of equipment. When using a microscope, you must:

1. Carry it upright with two hands with one hand holding the arm and the other hand supporting the base.

2. Use only the lens paper/Kimwipes and the solution provided to clean the lenses. Paper towels or cloths could scratch or leave lint on the lenses.

3. Begin with the low power (4X) objective when bringing an object into focus, and return the microscope to the cabinet with the low power (4X) objective in place. Also return the microscope with the stage in the lowest possible position.

4. Never use the coarse focus adjustment above low power.

5. When cleaning start cleaning lower the stage to the lowest position, then start with the 4x, then move to the 10X and finish with the 40X objective with one piece of lens paper soaked with lens cleaning solution. Use a separate piece of lens paper soaked with lens cleaning solution to clean the 100X oil immersion lens to make sure you don’t move oil from the 100X to the other lens.

How to Use the Microscope 

Watch the following two videos. When you get into a lab the instructor will show you their preferred way to handle, focus and clean a microscope.

Properties of the Microscope 

A. Magnification

The magnification of an image occurs by bending light through a series of lenses. Note that it is the image of the specimen that is magnified, not the specimen itself. The total magnification of the image is the product of the magnifications of the objective lens and of the ocular lens, as shown in this equation: 

total magnification = objective lens magnification x ocular lens magnification

Record the details about the objective lenses and the ocular lenses using information provided in the picture below. Use the equation above to determine the total magnification when each objective lens is in place, and enter the results in the table that follows:

Images of objective and ocular lenses

Power 

Stripe color of objective lens 

Magnification of objective lens 

Magnification of ocular lens 

Total magnification 

low 

 

 

 

 

middle 

 

 

 

 

high dry 

 

 

 

 

oil immersion 

 

 

 

 

Please fill out this table in your Post Lab Assignment (at the end of the lab) and submit it on Blackboard.

Measuring Objects with the Microscope

Here is a video that you might find helpful to solve for the diameter of the field at a new magnification using M1D1 = M2D2. You will also learn to estimate the size of an object under the microscope.

https://edpuzzle.com/media/5ef274d8cb1f783f21e007fc

Please fill out the table on your worksheet (at the end of the lab) calculating the new field diameters and submit the completed worksheet as an assignment.

The
field of view
(FOV) refers to the total area that is visible through the microscope. The field of view is circular. The field of view gets smaller as the magnification increases. It is possible to measure the
diameter of the field of view
, and that measurement can then be used to construct reasonable estimates of the size of any specimen contained within the field of view. But before we begin, we need to review the metric system, which is the system used in science to measure length, volumes, weights, and temperatures. 

  Remember that 1 mm = 1000 m 

Objects viewed with the light microscope are typically measured in
micrometers (m).
 

Determining the Diameter of the Field of View at 40x (low power) 

Observe the millimeter side of a ruler as it appears when placed across the diameter of the field of view on low power:  

 

Using the picture, estimate the length of the diameter of the field of view to the nearest half of a millimeter and enter it on your worksheet: 

Diameter of the field of view at 40x (low power): ______ mm 

Convert the diameter of field to micrometers (μm): _____ μm 

There is an
inverse
relationship between the total magnification and the diameter of the field of view—as magnification
increases
, the diameter of the field
decreases
in proportion. Thus, the diameter of field of view at any other magnification can be calculated mathematically according to the formula:

You will now need to fill out the same table in your Post Lab Assignment.
 

Power 

Total magnification 

low 

 

middle 

 

 

high dry 

 

 

oil immersion 

 

 

Diameter of field
	40x

Estimation of the Size of an Object under the Microscope:

The size of a specimen can be estimated by comparing it to the known diameter of the field of view (FOV) at any useful magnification. To do this, the object to be measured must be totally contained within the field of view. Let’s try measuring the length of a flea. 

Observe the image of a flea under low power (40X) and at medium power (100X) total magnification.

Low Power (40X) Medium Power (100X)

Approach A: The Percentage Method

Estimate the percentage of the diameter of the FOV occupied by the flea, and multiply that by the diameter of the FOV.

object size = percentage of diameter of FOV occupied x diameter of FOV

For example, if an object occupies 20% of a FOV that is 5,000 μm in diameter, the size of the object is: 0.20 × 5,000 μm = 1000 μm.

Approach B: The Counting Method

If there are many of the same objects along the diameter of the FOV (e.g. Elodea), estimate the size of one object by counting the number of objects. If estimating the size of a single object (e.g. an amoeba or a chick cell), imagine the number of objects that could be placed across the FOV. With either example, divide the diameter of the FOV by the counted or estimated number.

object size = diameter of FOV ÷ number of objects across the diameter of FOV

For example: If it seems 5 of the same object could be placed across the FOV, using this method, the size would then be: 5,000 μm ÷ 5 = 1000 μm. 

Estimate the size of the flea by using one of the methods described on the previous page. Try it for both magnifications. Include both of these calculations in your worksheet.

Estimated length of the flea at 40X: ____________________ m

Estimated length of the flea at 100X ____________________ m

Observing Living Cells by Preparing a Wet Mount of a Biological Specimen

A wet mount is a preparation of fresh material in a drop of water or other aqueous liquid (e.g., stain) on a glass slide.

A.
Elodea Leaf Cells

· Watch the following video showing a wet mount of Elodea leaf cells under a light microscope. The leaf is two cell layers thick. The cells are rectangular in shape and have oval green structures within them called chloroplasts. Some of these green structures are in motion, streaming around inside the cell. This phenomenon is called
cytoplasmic streaming
. Collectively, the cells look like a brick wall, with each cell resembling a translucent brick.

https://youtube.com/watch?v=0WCWgMqouaI%3Frel%3D0

· View and compare the 3 images of the Elodea leaf at increasing magnifications. Notice how the field of view decreases while the resolution increases.

· Draw three or four cells at high power magnification (400X) and label the cell wall, chloroplast, and cytoplasm of at least one of the cells. Include your calculations for estimating the size of an Elodea cell in the Post Lab Assignment at the end.

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Elodea leaf at 40X total magnification

Elodea leaf at 100X total magnification

Elodea leaf at 400X total magnification

Upload a photo of your drawings with labels and calculations in the Post Lab assignment.

B. Smear of Human Cheek Epithelial Cells

· Observe the image below of a wet mount of human cheek cells in methylene blue dye under high power (400x) magnification. The cells are called squamous epithelial cells and normally form a dense, tightly packed multiple layer of cells to protect the lining of the mouth cavity. However, the cells you are viewing appear scattered about or in small clumps because they were scraped off the lining.

·
Label the cell membrane, cytoplasm, and nucleus of at least one of the cells in the image below. Include your calculations for estimating the size of a cheek cell.

· Upload a photo of your drawings with labels and calculations in the Post Lab assignment.

Human cheek epithelial cells at 400X total magnification

The drawings (scans or photos of them) along with the calculations will need to be included in your post-lab assignment.

MICROSCOPE POST LAB ASSIGNMENT

Fill in the tables, answer the questions and attach the photos of your drawings to the document and upload your assignment on Blackboard.

total magnification = objective lens magnification x ocular lens magnification 

1. Record the details about the objective lenses and the ocular lens using information provided in the pictures. Use the equation above to determine the total magnification when each objective lens is in place, and enter the results in the table below: 

Power 

Stripe color of objective lens 

Magnification of objective lens 

Magnification of ocular lens 

Total magnification 

low 

 

 

 

 

middle 

 

 

 

 

high dry 

 

 

 

 

 

 

 

 

	oil immersion

 
2. Using the same total magnifications from the table above calculate the field size (in micrometers, µm) at each magnification. You will then use these field diameters to estimate the size of the flea, the Elodea cell and the cheek cell. 

Power 

Total magnification 

low 

40x 

 

middle 

 

 

high dry 

 

 

oil immersion

 

 

Diameter of field (µm)

3. Estimation of flea size at 40X:______________

4. Estimation of flea size at 100X:_____________

5. How well did your calculations match?

6. Which field size was easier to use in this case and why?

7. Please attach or upload your photo(s) of your drawings of the Elodea and Cheek cells at 400X total magnification. Be sure you have labeled your drawings appropriately, including the calculations for an estimate of the size of each of the cell types.

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