The Basics of Microscopy

Understand what is meant by the term "depth of field." How was this observed in the cork and threads/fibers slides?

-Distance through which you can move the specimen and still have it remain in focus. -The thread exercise; to determine the relative position of all 3 colored threads, you must raise and lower your objective lens slowly (aka "focusing up and down") and observe the order in which the threads go in and out of focus. As your objective lens approaches the slide, the first thread to come into focus is on the top.

Explain why a blue filter is used beneath the condenser of our microscopes. (Blue light is shorter wavelengths.)

-Increases contrast between a specimen and its background. -Better, higher resolution.

List rules for the proper care of your microscope. Know the checklist for putting a microscope away.

1. Carry microscope to workstation using both hands (one hand grasping the microscope's handle and the other supporting the microscope beneath its base) 2. Always begin focusing with the 4X objective 3. Use the course focus only with the 4X objective in place 4. Used immersion oil only with 100 X objective oil immersion lens in place 5. Use only one drop of oil (clean slide w lens paper after) 6. Lower the stage and then remove the slide when you are done 7. Always clean the microscope when you are done. Use a Kimwipe or lens paper and the alcohol in the labeled jars 8. Always place the 4X objective over the stage and be sure the stage is at its lowest position before putting the microscope away 9. Always turn off the light before putting the microscope away 10. Always wrap the cord correctly before putting the microscope away 11. Always return the microscope to the correct cabinet 12. Always place the ocular's towards the back of the cabinet Summary of putting away: Remove the slide and return it to its proper place. Clean any oil off the 100 X objective. Be sure the other objectives are clean. Turn off the light. With the cord up on itself, secure it with a Velcro tie, and let it dangle freely. Replace the cover. Return the scope to its cabinet with the ocular is facing the back.

Describe the change in the size of the field of view as magnification increases. Understand what that means in practical terms as one increases magnification when viewing a slide.

As magnification increases and the size of view decreases. As magnification increases, the diameter of the field of view decreases. You can see less area of the specimen as you increase the magnification.

Describe the change in working distance as magnification increases. What does this mean in practical terms with regard to observing samples at higher magnification?

As magnification increases, the working distance decreases. Working distance is how much space exists between the objective lens and the specimen on the slide. As you increase the magnification by changing to a higher power lens, the working distance decreases and you will see a much smaller slice of the specimen.

Compare the size of human blood cells with yeast cells and typical bacilli and cocci.

Human blood cells: ~12 nanometers Yeast: ~3-4 nanometers Bacilli: ~0.2-2.0 nanometers Cocci: ~1.0-2.0 nanometers

Evaluate the advantage to the microscopist of parfocality. (What does parfocal mean?)

Parfocal: having corresponding focal points all in the same plane; having sets of objectives or eyepieces so mounted that they may be interchanged without varying the focus of the instrument. Advantage: allows more accurate focusing at maximum focal length and then zooming back to a shorter focal length to compose the image.

Explain how the use of immersion oil allows the microscopist to obtain greater resolution.

Placing a drop of oil with same refractive index as glass between the cover slip and objective lens eliminates two refractive surfaces, can achieve greater magnification while preserving resolution (resolving power). Oil causes a better resolution because the refractive index of oil is very similar to refractive index of glass; it increases better magnification by eliminating the transfer from glass to air.

Describe the relationship between resolving power, numerical aperture, and the wavelength of light.

Resolving power: actual measurement of how far apart two points must be for microscope to view them as separate. Numerical aperture: a measure of a lens ability to capture light coming in from specimen and use to make an image. The smallest point that can be resolved by an objective is in proportion to the wavelength of the light being gathered, divided by the numerical aperture number.

Distinguish between a simple microscope and a compound microscope.

Simple microscope: consists of a single lens to enlarge an object. Compound microscope: includes an eyepiece and one or more objectives where the eyepiece enlarges the resolved image created by the objectives.

Recognize how the optics of the microscope change the orientation of the image seen when compared to the orientation on the slide.

The optics of microscope inverts the image (upside down). (example was the letter "e" slide)

Understand how to properly clean the optical components of a compound microscope.

To clean microscope eyepiece lenses, breathe condensation onto them and then wipe them with lens tissue. Kimwipes are made by Kleenex and generally will work well. For stubborn spots wipe the surface with tissue moistened with 95% alcohol. Wipe the lens dry with a dry tissue.

Given the magnifications of an ocular and an objective lens, calculate the total magnification of a compound microscope.

Total magnification= magnification by the objective lens X magnification by the ocular lens

Locate and name the parts of your microscope; explain the function of each part.

pg. 142 in the lab manual