1.2: Microscopes
Many biological specimens such as cells and tissues are difficult or impossible to be seen with the naked eye so they must be magnified to be studied. Consequently, the basic operation and care of microscopes is an important skill in biology.
Two basic types of microscopes are used in our introductory biology labs: compound light microscopes and stereo microscopes (aka dissecting microscopes).
Compound light microscopes pass visible light through two sets of magnifying lenses (the ocular and the objective lenses) to magnify specimens mounted on a glass slide and placed on the flat surface ( stage ) of the microscope. The specimens must be sliced very thin in order for light coming from the light source to pass up through them so that they can be viewed. Specimens are sometimes stained with dyes to add contrast and to make structures more easily identifiable. Microscopic examination of cells and tissues allows students to observe the principle of complementarity - how cell and tissue structure determines function.
Exercise 1: Parts of the Microscope
In this exercise, you will identify and learn the functions of various microscope parts. Proper practice for handling and use of compound light microscopes is as follows:
Materials:
- Compound light microscope
Procedure:
- Obtain a compound light microscope as directed by your instructor.
- Carry the microscope upright with one hand supporting the base and the other hand grasping the arm. Care should be taken not to bump the microscope on the microscope cabinet, chairs, tables, or other obstacles. Gently place your microscope on your laboratory bench and remove its protective plastic cover (if present).
- Do not push or slide the microscope across the table. This causes vibrations that can loosen screws or misalign microscope parts.
- Clean the lenses with lens paper (if needed). The lenses on the microscope scratch easily. If you need to clean them, use ONLY lens paper (KIM Wipes are not lens paper).
- Before you use the microscope, locate the parts of the microscope and label Figure \(\PageIndex{1}\) . Additionally, Table 1 provides the function of each part.
|
Microscope Part |
Function |
|---|---|
|
Head |
Upper part of microscope that extends from arm and contains the ocular lenses and revolving nosepiece with objective lenses. |
|
Arm |
Supports the body tube and lenses. Use the arm and base to carry the microscope. |
|
Base |
Supports the entire microscope. |
|
Ocular (eyepiece) |
The lens in the upper part of the microscope. Monocular microscopes have one ocular, while binocular microscopes have two oculars. Ocular magnification is 10x . |
|
Body tube (turret) |
Holds the ocular at one end and the nosepiece at the other. Conducts light rays. |
|
Revolving nosepiece |
Located at the lower end of the body tube. A revolving device that holds the objective lenses ( aka objectives). |
|
Objective lenses |
Located on the revolving nosepiece. Each lens has a different magnifying power. The smallest objective ( scanning objective ) is the smallest magnification at 4x, followed by the low power objective at 10x, the high power objective at 40x, and the highest magnification (immersion oil objective) at 100x. Only one objective may be used at a time. The selected lens is rotated into position by turning the nosepiece. |
|
Stage |
The horizontal platform upon which the slide rests. |
|
Condenser |
Lens beneath the stage that concentrates and focuses the light before it passes through the specimen to be viewed. |
|
Iris diaphragm lever |
Small lever beneath the condenser which controls the amount of light passing through the condenser. |
|
Light source |
Provides a beam of visible light to be passed through the specimen. |
|
Mechanical stage |
Moveable part of the stage controlled by stage adjustment dials located below the stage. Allows the observer to move the stage forwards, backwards, left, and right. |
|
Coarse adjustment knob |
Located on either side of the arm. Moves the stage to bring the object into focus. This knob should only be used when the scanning (4x) objective is positioned above the stage! |
|
Fine adjustment knob |
Located within the coarse adjustment knob. Allows fine focusing of the specimen. |
- The head of the microscope supports the first set of magnifying lenses called the ocular lenses . These are located at the top of the microscope. These lenses are where we place our eyes and are also referred to as eyepieces. The ocular lenses magnify a specimen ten times (10x). If your microscope is binocular, the interpupillary distance (the distance between the eyepieces) can be adjusted to accommodate this distance.
- The second set of four lenses is located on a revolving nosepiece . Collectively known as objective lenses , these lenses can magnify a specimen four times (4x), ten times (10x), forty times (40x), or 100 times (100x), respectively. Magnification values are etched on the sides of the metal casings that protect the lenses.
- Closely related to the topic of magnification is resolution. Resolution (or resolving power ) is the ability of a microscope to distinguish two adjacent structures as distinct, or separate. The higher the resolution, the better the clarity and detail of the image.
- The bending of light through objective and ocular lenses is called refraction. This is what can give us our magnification power. At even higher magnifications, excessive refraction can cause distortion of the image but the oil immersion objective lens (100x) can remedy this problem by eliminating the gap between the specimen and the objective lens with a drop of oil. We will NOT be using the oil immersion lens in this class unless otherwise noted by your instructor.
- The light source in these microscopes can be controlled at a variety of points on the microscope, which you will see as part of this lab. Adjusting the light source correctly can be as important to see your image as adjusting focus.
- Calculating Magnification: When using a compound light microscope, both the ocular lens and objective lens help magnify the image. Therefore, to calculate the total magnification of an image, the contribution of each lens must be taken into consideration. Total magnification can be calculated using a simple formula:
\[\text{Total magnification} = \text{Ocular lens power} \times \text{Objective lens power}\]
Calculate total magnification values for your microscope and record the values in Table 2 below.
- Always begin viewing a slide using the scanning (4x) objective . Never begin an observation with the higher-powered (10x, 40x, 100x) objectives. Doing so could result in broken slides or scratched lenses.
- Never use the coarse focus adjustment at high magnification. Once a specimen is brought into focus using the lowest power, you can rotate to a higher power objective lens to increase the magnification. You will probably need to focus only slightly, as most light microscopes are parfocal , meaning that the image remains nearly in focus as you change lenses (from lowest to highest power).
- Replace the microscope properly. When you are finished using the microscope, turn off the light, remove the last slide from the stage, and wipe any material from the stage. Lower the stage and move the lowest power (4x) objective into position. Bundle the electrical cord securely (not around the arm of the microscope), replace the plastic cover, and put the microscope back in the storage cabinet.
Exercise 2: Focusing & Image Inversion
The optics of a light microscope’s lenses change the orientation of the image the user sees. A specimen that is right-side up and facing right on the microscope slide will appear upside-down and facing left when viewed through a microscope, and vice versa. Similarly, if the slide is moved left while looking through the microscope, it will appear to move right. If moved down, it will seem to move up. This occurs because microscopes use two sets of lenses to magnify the image. Because of the manner by which light travels through the lenses, this system of two lenses produces an inverted image.
You can get a slide nearly into focus without ever looking into the ocular lens by following these simple steps:
- Turn the light on and adjust the objective lens to the lowest power scanning lens.
- With the light on, place your slide on the stage and center the specimen in the light using the stage adjustment knobs.
- Raise the stage the whole way up, then bring it down with half a rotation of the coarse adjustment knob.
- Compound light microscope
- Prepared slides: letter “e”
Procedure:
- Obtain a slide of the letter “e.”
- Look at the letter “e” and draw what you see in the circle provided.
Draw the letter “e” as seen on the slide while holding it in your hand.
- Place the slide on the stage . Secure it with the stage clips . Make sure that the scanning objective (4x) is clicked into place. Use the stage adjustment knobs to center the letter “e” over the light source and directly in the center of the field of view (the lighted circular area you see as you look through the ocular lenses).
- While looking into the ocular lenses , use the coarse focus adjustment to bring the letter “e” into focus. Use the fine focus adjustment to “fine tune” the image. Try adjusting the light with the iris diaphragm lever. Decreasing the aperture size decreases the amount of light on the specimen and increases contrast.
- Observe the position of the letter “e” as it appears in the field of view (the circular area that can be seen when looking through the ocular). Draw the letter as it appears through the microscope in the circle provided.
Letter “e” as seen through microscope
- Compare your drawing from step (b) to that of step (e). How do the two drawings differ? __________________________________________________________
- While observing the letter “e” through the ocular lenses, describe the movement that you observe as you move the slide:
- to the left - _____________________________________
- to the right - ____________________________________
- forward - ______________________________________
- backward - _____________________________________
- When you view the letter “e” using the scanning objective , what is the total magnification ? _________________________________________
- Center the letter “e” in the field of view. Look at your microscope from the side (not through the oculars) and rotate the revolving nosepiece so the low power (10x ) objective clicks into place.
- Focus the letter using the fine focus adjustment. (Do not use the coarse focus adjustment.) Most microscopes are parfocal . This means that when you focus the specimen under the lowest magnification, that the specimen is “almost” in focus at any higher magnification, such that you will only need to use the fine focus adjustment to focus the image.
- Under low power , how many times has the letter “e” been magnified? __________________________________________________________
- Compared to scanning magnification , does the letter appear larger? __________________________________________________________
- Compared to scanning magnification, can you see more detail in the letter or the paper it is printed on? ___________________________________
Exercise 3: Diameter of the Field of View
Since the scanning objective is 4x and the low power objective is 10x, images will be magnified more with low power than with scanning power. Because objects will appear larger, the low power “field of view” will be smaller than the scanning power field . Therefore the relationship between the diameter of the field of view and the magnification is inversely proportional. Not only does measuring the field of view at different magnifications demonstrate this property, but once you know the diameter of the field of view in millimeters (mm) at various magnifications, you will be able to estimate the size of the cells or other structures being viewed.
- Compound light microscope
- Clear metric ruler
Procedure:
- Lower the stage, and bring the scanning objective (4x) back to the center position. Remove the letter “e” slide if you haven’t already removed it.
- Position a clear plastic ruler across the stage, and focus on its edge, so that the edge of the ruler, with millimeter markings, is visible in the field of view and equally bisects the field of view.
- The distance between two lines on the ruler is 1mm. Draw what you see in the circle provided. Measure the diameter of the field of view (in mm). ________ mm.
- Leaving the ruler in place, rotate the low power (10x) objective into position.
- Use the fine focus if necessary to bring the ruler into focus.
- Estimate the number of millimeters that you see in the field of view:
_______mm
- Leaving the ruler in place, rotate the high power (40x) objective into position.
- Use the fine focus if necessary to bring the ruler into focus.
- Estimate the number of millimeters that you see in the field of view:
_______mm
- What happens to the field of view when you increase magnification? Explain. _________________________________________________
- Do you see MORE or LESS of an object if you increase magnification? Explain. _________________________________________________
- Move the scanning (4x) objective lens back into place, lower the stage with the coarse adjustment knob, and remove the ruler.
- Place the letter “e” slide back onto the stage and secure it with the stage clip. Using the scanning objective lens, estimate the diameter of the letter “e”. If it occupies ½ of the field of view, then it is ½ times the measurement you recorded in step (c) above. Record the diameter of the letter “e”. _____________________ mm
Exercise 4: Depth of field
Depth of field is the area (top to bottom) of an object that comes into focus while slowly moving the fine adjustment knob up and down. Because the depth of focus is very short in the compound microscope, you must focus up and down to clearly view all of the planes of a specimen.
- Compound light microscope
- Slide of crossed colored threads
Procedure:
- Obtain a slide with colored threads mounted together. Place it on the stage and focus using the scanning (4x) objective . The center of your field should be the point where the three fibers cross each other.
- Focus up and down using your coarse focus adjustment . Under scanning magnification, are all three fibers in focus at the same time? ______________________________________________________
- Can you easily tell which fiber is on top and which is on the bottom? ______________________________________________________
- Rotate the nosepiece so that the low power (10x) objective clicks into place.
- Focus up and down using your fine focus adjustment . Under low magnification, are all three fibers in focus at the same time? ______________________________________________________
- Can you easily tell which fiber is on top and which is on the bottom? ______________________________________________________
- At which magnification is there a greater depth of field ? ______________________________________________________
- Use the iris diaphragm to change the amount of light passing across the fibers. Note how changing the position of the diaphragm helps to increase (or decrease) your ability to view objects.
- Does the 4x or the 10x objective have a shorter depth of field? _____________________________________________________
- Using the fine focus adjustment, slowly rotate the knob until all three threads are just out of focus. Slowly refocus using the fine focus adjustment.
- Which thread comes into focus first? _______________________
- Is this thread lying under or over the next thread? _____________
- Slowly rotate the high power (40x) objective into position and focus up and down using the fine focus adjustment only. Does the 10x or the 40x objective have a shorter depth of field? ___________________________
Exercise 5: Wet Mount of Human Epithelial Cell (Cheek Cell)
According to the cell theory, the cell is the fundamental biological unit, the smallest and simplest biological structure which possesses all of the traits of something that is living. All living organisms are composed of one or more cells, and every activity taking place in any living organism is ultimately related to the metabolic processes that are taking place in cells. Therefore, to understand the processes of life, it is necessary to understand the structure and function of the cell. Cells that line the interior of the mouth and cheeks are situated very close together, similar to tiles on a floor. These thin cells form a thick layer that protects the underlying tissue from abrasion and foreign pathogens (e.g. viruses and bacteria). The cells comprising the most superficial layer are continually sloughed off and replaced by underlying cells. Gently scraping the lining of the cheek removes the superficial cells. In this activity, you will prepare a wet mount slide of cheek cells and observe them under the compound light microscope.
- Compound light microscope
- Clean microscope slides and coverslips
- Toothpicks
- Methylene blue dye
- Water with dropper
Procedure:
- Obtain a clean microscope slide and coverslip. Add one drop of water to the slide.
- Obtain a flat toothpick and obtain a sample of your cheek epithelial cells from the inside lining of the oral cavity. The goal is to gently scrape loose cells free, not to draw blood from your cheek.
- Apply the cells to the slide, rotating the toothpick between your thumb and forefinger to dislodge the cells into the water. Dispose of the toothpick in the biohazard trash.
- Obtain a small bottle of methylene blue dye. Make sure that the dropper does not actually touch the slide ( do not contaminate the dropper bottle with your cheek cells ). Let a VERY small drop of methylene blue dye fall onto the slide. (*Methylene blue will stain just about anything it touches a deep blue, so be careful not to get it on your skin or clothing.)
- What is the purpose of adding stain to the biological specimen? __________________________________________________
- Place a clear cover slip on top of the specimen. You have just prepared a
wet mount slide !
- Bring the epithelial cells into focus using the scanning (4x) objective and the coarse focus adjustment. Zoom in on a few cells by switching to the low power (10x) objective lens.
- Confirm with your instructor that you have viewed a cheek epithelial cell.
- Switch the objective lens to high power (40x) . Sketch one cell in the circle below.
Total Magnification_______
- What is the genetic material found in the nucleus of these eukaryotic cells? _________________________________________________
- What is the approximate size of one cell? ___________________ mm
- How do you know this? ____________________________________
Exercise 6: Observation of Plant Cells (Elodea Cells)
Leaves of Elodea, a common aquatic water plant, are great for observing the major characteristics of a typical plant cell. In this activity you will prepare a wet mount and examine one of the leaves from the Elodea under the compound light microscope.
- Compound light microscope
- Clean microscope slides and coverslips
- Elodea cells
- Water with dropper
- Optional: Onion epithelial cells or prepared slide of onion root tip
- Methylene blue dye (only if using onion epithelial cells as dye is not needed to see the cellular structures in the Elodea leaf)
Procedure for Elodea Cells:
- Obtain a clean microscope slide and coverslip.
- Using forceps, remove an Elodea leaf (smallest, youngest leaves are best) from the sprig of Elodea provided and place it on the slide. Make sure it is pressed down and flat.
- Add a drop of water to cover the top of the leaf.
- Place a clear cover slip on top of the specimen. You have just prepared a
wet mount slide !
- Bring the plant cells into focus using the scanning (4x) objective and the coarse focus adjustment. Zoom in on a few cells by switching to the low power (10x) objective lens.
- Switch the objective lens to high power (40x) . Sketch several Elodea cells in the circle below.
Total Magnification_______
- Identify the following structures:
- Cell wall - the rigid framework consisting of the carbohydrate cellulose that surrounds the cell and lies outside the cell membrane. Its function is to give the cell a definite shape and provide support. Cell walls are not found in animal cells.
- Protoplasm - the organized contents of the cell, exclusive of the cell wall.
- Central vacuole - the membrane bound sac, filled with water and dissolved substances, that lies within the cytoplasm. Its functions include the storage of metabolic wastes as well as providing turgor pressure which gives the cells support.
- Cytoplasm - the protoplasm of the cell exclusive of the nucleus.
- Chloroplasts - the green spherical organelles containing the pigment chlorophyll that is involved in photosynthesis. As the microscope light heats up the water on the slide, you may observe the cytoplasm and the chloroplasts moving around the central vacuole in a process called cytoplasmic streaming .
- Nucleus - the organelle within the cytoplasm that contains the genetic information in the form of DNA. Since dye has not been used to visualize cellular structures, the nucleus may be transparent and difficult to observe.
- What three structures are observed in the Elodea cells that are unique to plants and not observed in animal cells ? ___________________________________________________________
Procedure for Onion Epithelial Cells: (Optional)
- Remove the thin, transparent epidermis (skin) from an onion leaf to prepare a wet mount slide. Alternatively, you may view a prepared slide of onion root tip. Do not discard commercially prepared slides.
- Place on a clean slide and add a drop of methylene blue. Do not contaminate the dropper (do not touch the onion skin with the dropper). Cover with a clear coverslip.
- Observe with the scanning objective lens using the coarse focus adjustment first, then the fine adjustment knob.
- Observe using the low power objective lens. Make sure you see the rectangular shaped onion cells. Confirm with the instructor, if necessary. Sketch the onion cells in the circle below. On your drawing label the cell wall (lacking in animal cells), cell membrane, cytoplasm, central vacuole, and nucleus. Be sure to indicate the total magnification used in your drawing.
Sketch onion cells
Total Mag. ____
- Estimate the size of one cell: ____________________________ mm
Exercise 7: Stereo Microscope
Stereo microscopes (also called dissecting microscopes; Figure 2 ) also contain 2 sets of lenses (ocular and objective lenses). The ocular lenses on a stereo microscope, like a compound light microscope, magnify by a factor of 10x. The objective lenses, however, have relatively low magnification. There is a great deal of variation in stereo microscopes and the manner in which they achieve higher magnification. Stereo microscopes include an additional magnification system that makes the final image appear to be upright.
They are used for viewing and manipulating relatively large specimens which can be viewed in three dimensions. They have a binocular feature that creates a stereoscopic effect. They can be used to study entire small organisms since their depth of field is much greater than the compound light microscope. The light source can be directed down onto a specimen (reflected light) as well as up through the specimen (transmitted light), which permits the viewing of objects too thick to allow for the transmission of light. It is also possible to view multiple samples in a petri dish placed on the microscope’s stage with light that is projected from below.
- Stereo microscopes
- Variety of objects to be viewed (e.g. feather, shell, penny, preserved insects, crystals)
Procedure:
- Your instructor will describe how to use the stereomicroscope.
- Use the stereomicroscope to view the variety of materials provided for you.
Make a drawing of 2 of the items you viewed with the dissecting microscope.
Exercise 8: Electron Microscope
Electron microscopes, first developed in the 1940s, use a beam of electrons instead of light to magnify an object. They can magnify objects as small as 2 nanometers (0.00000004 inches) over 100,000 times.
Transmission electron microscopes are used to study internal cell structure and are analogous to compound light microscopes in that regard. Specimens are cut into thin sections, usually “stained” with heavy metal atoms (atoms with large atomic numbers) that attach to cell structures. An electron beam is then focused through the specimen. Figure 3 is an example of the type of image an electron microscope can produce.
Scanning electron microscopes , analogous to stereo microscopes, allow a specimen’s surfaces to be observed in detail. The object is chemically frozen and then coated with a thin film of metal. An electron beam excites surface electrons on the specimen and produces a three-dimensional image. Figure 4 is an example image that is produced by an SEM.
Electron microscopes are expensive and require special training. Even though the preparation techniques used to prepare specimens for electron microscopy kill most living cells, some cells such as Tardigrades can withstand these harsh treatments.
Questions For Review:
- Why, specifically, did we look at the letter “e” slide, threads slide, and the ruler?
- What cellular parts of your cheek cell could you observe?
- What does it mean if a microscope is parfocal?
- What is the relationship between the magnification and the diameter of the field of view? Explain.
- What two parts of the microscope do you use to carry it?
Practical Challenge Questions:
- Imagine you are looking at a slide on high magnification and you lose focus on it. What should you do?
- You are using a microscope you haven't used before and you notice the microscope has a 10x ocular as well as a 73x objective lens. What will the total magnification be if this objective lens is positioned over the stage?
- You are given several specimens to observe with a microscope but you are unsure what type of scope to use. What are some reasons that you may choose different microscopes?
- Why is the resolving power of the transmission electron microscope so much greater than that of the compound light microscope?
References
Belwood, Jacqueline; Rogers, Brandy; and Christian, Jason, Foundations of Biology Lab Manual (Georgia Highlands College). “Lab 3: Microscopy,” (2019). Biological Sciences Open Textbooks . 18. CC-BY https://oer.galileo.usg.edu/biology-textbooks/18