Microscopy Flashcards

Compound Light Microscope and Oil Immersion Microscopy

Learning Objectives

• Explain the function associated with the parts of the microscope.
• Explain the role magnification and resolution play in brightfield microscopy.
• Describe how oil-immersion increases the resolution for a specimen.
• Use the microscope to identify a specimen’s morphology (and arrangement in the case of bacteria).
• Calculate total magnification.
• Understand how the orientation and movement of the specimen’s image changes when viewed though a compound light microscope.
• Demonstrate how to use oil immersion with the 100x objective for bacterial slides.

Background

• Microscopes are essential tools in biology, enabling the visualization of tiny objects.
• The term microscope translates to “to view the tiny”.
• The lab utilizes a compound light microscope.
• Light microscopes use light and lenses to magnify the specimen's image.
• "Compound" indicates that light passes through the specimen and then through two different lenses.
• Objective lens: The lens closest to the specimen.
• Ocular lens (eyepiece): The lens nearest to the user’s eye.
• Specimens are placed on glass slides.
• Types of slides:
  • Prepared slide: Permanent, purchased from a science supply company.
  • Temporary slide: Fixed and stained in the lab for short-term use.
  • Wet mount: Made fresh in the lab for temporary use.
• The objective lens is primarily responsible for magnifying the specimen, typically ranging from 4x to 100x.
• Total magnification is calculated by multiplying the magnification power of the objective lens by the magnification power of the ocular lens (usually 10x).
• Formula for total magnification: Total Magnification = (Objective Lens Magnification) \times (Ocular Lens Magnification)
• Example: A 40x objective lens with a 10x ocular lens results in a total magnification of 400x.

Light and Magnification

• Light Microscopes rely on light for illuminating samples, requiring appropriate light passage through objective lens.
• As magnification increases, the amount of light reaching the ocular lens decreases due to smaller numerical apertures capturing less light.
• The field of view becomes smaller which reduces overall brightness.
• Increasing the iris diaphragm opening helps increase light intensity with higher magnification objectives.

Oil Immersion Microscopy

• The 100x objective lens is used to view very small objects, such as bacteria.
• Image quality decreases at high magnification, appearing fuzzy or out of focus due to light refraction.
• Light is bent by the glass slide, refracting away from the objective, which makes the specimen appear out of focus.
• Immersion oil is used to bridge the gap between the slide and the objective, allowing more light to travel into the objective by preventing refraction.
• Refractive index: A measure of how well light passes through a substance.
• The refractive index of oil is similar to glass, acting like liquid glass.
• This results in a better, more focused image.

Basic Procedure for Brightfield Light Microscopy

1. Set-up
   • Microscope is plugged in and the light/power switch is turned on.
   • Stage in lowest down position.
   • 4x scanning objective in place.
   • Microscope slide properly set in stage clips.
   • Move microscope slide so light is passing through specimen (is centered over the light).
2. Visualize the specimen with the scanning (4x) objective
   • Raise the stage using the course adjustment knob.
   • Once the specimen is in focus, move the stage so that the specimen is in the center of the field-of-view (the circle of light you see in the ocular).
   • Adjust the light intensity as needed.
3. Move the low power objective (10x) lens into place
   • Touch nothing - look through the ocular lens; the sample should mostly be in focus already because of the parfocal properties of the microscope.
   • If needed, ONLY use the fine adjustment knob to adjust the stage.
   • Adjust light intensity as needed.
4. Move the high power objective (40x) lens into place
   • Touch nothing - look through the ocular lens.
   • If needed, ONLY use the fine adjustment knob to adjust the stage.
   • Adjust light intensity as needed.
5. Add a small drop of immersion oil to the specimen, and immediately move the 100x oil objective into place
   • Only a small amount of oil is needed.
   • The oil will touch both the slide and the objective, making a “bridge” between the two.
   • Touch nothing - look through the ocular lens.
   • If needed, only use the fine adjustment knob to adjust the stage.
   • Adjust light intensity as needed.

• If the specimen is lost at any point, return to Step 1 and repeat.

Troubleshooting and Good Practices

• Specimen is dark: Increase light using the rheostat or adjust the diaphragm.
• Debris in the field of view: Clean the slide, ocular, and objective lens with lens paper.
• Background light is too bright: Turn down the rheostat.
• Always carry the microscope upright with two hands, holding the arm and the base.
• If you cannot locate the specimen on scanning, try the low power objective (common with bacterial samples).
• Be mindful of the distance between the objective lens and the stage to avoid crashing the stage into the objective.
• The slide and the objective should nearly touch only with the oil immersion objective during oil immersion microscopy.

Cleaning of Equipment

• Slides: Remove the slide from the stage and wipe off the oil completely with a Kimwipe; use a little 70% ethanol (EtOH) if the slide appears “greasy”.
• 100x objective: Use lens paper to wipe off the oil (not a Kimwipe!). Dab a little 70% EtOH onto the lens paper to help remove the oil. Continue to wipe the 100x objective lens with the lens paper until all oil is removed.

References

• Modified from Dr. Timothy Rochbach’s “BIO211-Microscope-Lab Report” Fall 2024.
• Wikipedia: https://en.wikipedia.org/wiki/Oil_immersion
• Lab: Using a Compound Light Microscope: https://cpb-us-e1.wpmucdn.com/share.nanjing-school.com/dist/3/28/files/2013/02/using-microscope-lab-sq892v.pdf