Exercise 1: Review Magnification and Resolution
Fill in the following table:
| Magnification Objective Lens | Magnification Ocular Lens | Total Magnification | |
| Objective 1 | |||
| Objective 2 | |||
| Objective 3 | |||
| Objective 4(Oil Immersion) |
Fill in the table with the numerical aperture for each objective lens (simply look at the objectives on your microscope to find the NA for each). The objectives on your microscope are air objectives, except for the 100x, which is an oil immersion objective. Then, calculate the maximum resolution that can be obtained for each lens. Use λ=550nm (average visible light wavelength) and λ=400nm (close to the smallest wavelength detectable by the human eye).
Melissa Hardy, CC BY-SA 4.0
Resolution may be calculated as:
r = 0.61λ/NA
r = resolution = minimum distance between points
λ = wavelength
NA = numerical aperture (fixed attribute of a lens)
| Objective | Numerical Aperture (NA) | Maximum resolution (λ=550nm) | Maximum resolution (λ=400nm) |
| 10x | |||
| 40x | |||
| 63x | |||
| 100x |
- How does wavelength affect resolution? What happens to resolution as wavelength decreases?
(Note that, although you have calculated a theoretical maximum resolution, the actual resolution may be lower. Aberrations in the objective lenses decrease the actual resolution. Wavelength is also a consideration. The human eye is best at detecting green light, and fine detail may be undetectable to the eye when a much longer or shorter wavelength is used.)
Exercise 2: Read and initial these important microscope use guidelines
General Microscope Procedures
- Make sure all backpacks, purses, etc. are off the benchtop.
- Carry microscope by the base and arm with both hands.
- Place the microscope gently on the bench.
- Do not push or slide the microscope around. If it needs to be moved, pick it up and set it down carefully.
- When you are done with the microscope, lower the stage as far as possible and make sure that the lowest power objective (10x) is clicked into place.
- Never use paper towels or tissue to clean the microscope lenses! Only use lens paper with lens cleaner.
- Do not allow the objective to touch the slide. When switching objectives, turn the nosepiece slowly and watch carefully (with the naked eye, not through the eyepieces).
I have read and will follow these instructions: ____________________ (initial here)
Exercise 3: Set up Köhler Illumination
Setting up the microscope
Before viewing or photographing a specimen, it is important to set up your microscope correctly, including setting it for Köhler illumination. This will ensure that the resolution and contrast is as high as possible. Köhler illumination should be set up at the beginning of each lab session.
First, focus on a prepared slide and adjust the eyepieces:
- Clean the objective lenses with lens cleaner and lens paper.
- Plug your microscope in to power supply and turn on the illuminator.
- Place a prepared slide on the stage, using the stage clip to hold it securely in place.
- Use the coarse focus knob to focus. Always start with the stage as low as possible and using the lowest power objective (10x for most of our microscopes). Carefully bring the stage closer to the objective lens until you can see it through the eyepieces.
- Use the fine focus until you can see the image as sharply as possible.
- Move the mechanical stage until your focused image is also centered.
- Adjust the eyepieces until they are at the correct interpupillary distance for you. This will be different for each person. Move the eyepieces closer to each other or farther from each other until you see a single circle of light (not two!). You should be able to comfortably view the specimen with both eyes. If you are closing one eye, you need to adjust the eyepieces.
- If the microscope has a focusing eyepiece, make sure the focus is set correctly. To set the focus, first close the right eye and use the fine focus knob until the image is as sharp as possible for the left eye. Now close the left eye and open the right eye (looking through the focusing eyepiece). Rotate the focusing eyepiece (NOT the fine focus knob) until the image is as sharp as possible.
Next, set up Köhler illumination
- With a slide on the stage and in focus, open the condenser diaphragm all the way.
- Close the field diaphragm all the way. You should see a small circle of light in the center of your field of view.
- Adjust the condenser focus knob until the edges are sharp.
- If the circle of light is not centered, use the condenser adjusting screws to center it in your field of view.
- Open the field diaphragm until it is just large enough to be outside your field of view.
- Now adjust the condenser diaphragm by slowly closing the condenser diaphragm while looking through the eyepieces until the specimen darkens slightly.
(NOTE: this is a quick and dirty method which will achieve good illumination. The correct way to do this is to remove an eyepiece and look down the tube, then adjust the condenser diaphragm until it illuminates 65-80% of the diameter. However, we do not want you to remove eyepieces from the microscopes, to avoid getting dust in the ocular tubes).
Now, you are ready to observe specimens
- Once you’ve focused using the lower power objective, switch to the higher power objective (40x). Use the fine knob to refocus and move the mechanical stage to re-center your image. Your image must be in focus with the lower power objective, or you will probably not be able to find it with the higher objective.
Demonstrate microscope setup:
Once you have run through the steps above and are comfortable with them, have your instructor watch you set up the microscope, including Köhler illumination.
Instructor’s initials here: ________________
Exercise 4: Orientation of Images in the Microscope
A large part of the learning process of microscopy is getting used to the orientation of images viewed through the oculars as opposed to with the naked eye. A common mistake is moving the mechanical stage the wrong way to find the specimen. This procedure is merely practice designed to make new users more comfortable with using the microscope.
Materials
- Compound microscope
- Stereo microscope
- Microscope slide with the letter “e”
Procedure
- Place the letter “e” slide onto the mechanical stage. Place it so the “e” is facing you.
- Use the lowest power (10x) objective and course focus adjustment to focus, then move the mechanical stage around to find the letter “e”. Note the orientation when viewed through the oculars. Draw what you see below.
- When the stage moves left, which way does the image move? ______________
- When the stage moves away from you, which way does the image move? _______________
Does the lens of the microscope reverse the image? _________
Does it flip the image (upside down)? _________
- Now place the letter “e” slide onto the stereo microscope in the same orientation (so the “e” is facing you).
- Use the focus knob until the image is sharp. Draw what you see below.
Does the lens of the microscope reverse the image? _________
Does it flip the image (upside down)? _________
Exercise 5: Investigation of Pond Water and Microorganisms
Materials
- Compound microscope
- Microscope slide
- Coverslip
- Transfer pipette
- Pond water sample
- Brine shrimp (Artemia)
Procedure
- Using the transfer pipette, transfer a drop of pond water onto a microscope slide. The best specimens often come from the bottom and may contain chunks of algae or other debris that you can see with your naked eye.
- Slowly lower a coverslip onto the slide using either your hand or a dissecting needle.
Christine Anderson and Lisa Bartee, Biology 101A Lab Packet, 2016, CC BY 4.0
- Use the low power (10x) objective to focus, then move the mechanical stage around to scan the slide for live microorganisms. You are looking for tiny swimming things- they may look green or clear and might be very small. Choose one to focus on and center it in your visual field. You may wish to use ProtoSlo (methylcellulose) to keep your organisms from swimming too quickly.
- Switch to the next higher-powered objective (40x) and view your chosen organism. Use a higher objective if necessary.
- Repeat the procedure for the brine shrimp (Artemia).
Organism #1 Organism #2
Exercise 6: Take a picture of one of your organisms
- Take one of your wet mount slides to the compound microscopes that are equipped with cameras.
- Follow the instructions at the microscope station to take a picture of your organism.
- Center, crop, and add a scale bar.
- Rename the file to include your name, objective used, and specimen. For example, if I took a picture of a brine shrimp, I might name it: “Melissa Hardy brine shrimp 10x”
- Submit your picture via Canvas.
Cleanup
- Store microscope with the lowest power objective in place and the stage in its lowest position.
- Wrap cord around microscope.
- Replace slides in original slide tray.
BIOL 1625 