0.2: Overview of Microscopy

Parts of the Compound Light Microscope

• Ocular lenses (eyepieces): Our microscopes are binocular which means that there are two ocular lenses for you to look into. The specific microscopes that we have in the classroom are butterfly binocular lenses which means that you can adjust the distance between each of the lenses (similar to how a butterfly flaps its wings) to better match the distance between your eyes. Each has a magnification of 10x.
• Objective lenses: Four lenses mounted on the rotating nosepiece —
  • 4x (scanning power): This lens should always be the first that you use to “scan” the slide from a relatively low magnification until you see the area of the slide that you wish to focus in on.
  • 10x (low power): This lens will allow you to see larger microscopic animals and plants structures and can also be used as a step as you make your way to using the 40x/high power lens.
  • 40x (high power): This lens should be used when examining details of a specimen or some of the larger unicellular organisms we will observe.
  • 100x (oil immersion): This lens is used only with immersion oil for the observation of fine details as well as prokaryotes such as bacteria which tend to be much smaller than most eukaryotes. When using the oil immersion lens, you should go directly from 4x to the 100x without bringing the 10x or 40x lens near the oil that you have placed on the slide.
    • These lenses are parfocal, meaning once the specimen is focused under one objective, only minor adjustments are needed when switching to a higher power.
• Stage: The flat platform that holds the slide in place.
• Stage clip: This has a lever that you can use to secure your slide so it doesn’t move. You will notice that there are two notches inside this clip-one that accommodates the traditional glass slides and a smaller, notch that accommodates deep-welled slides.
• Stage adjustment knobs: These knobs can be found hanging from the right side of the stage and function to move the slide left/right and forward/backward for precise positioning.
• Coarse focus knob: This is the larger of the two knobs which moves the stage up and down quickly to bring the specimen into general focus.
• Fine focus knob: This knob is used to make small adjustments to sharpen the image once the specimen is visible.
• Iris diaphragm: This is a small lever that can be found just underneath the stage and it controls the amount of light passing through the specimen. Adjust to improve contrast and clarity.
• Light source: Provides illumination from below the stage.

   

Using the Compound Light Microscope

1. Set Up the Microscope
   • Obtain a microscope from the microscope cabinet using care to place a hand under the base as well as the arm of the microscope to carefully carry the microscope to your workstation.
   • Place the microscope on the lab bench, plug it in and turn on the light source.
2. Start with the Scanning Power Objective (4x)
   • Rotate the nosepiece until the 4x objective lens clicks into place.
   • Always start and end with this lens to protect the microscope and slides.
3. Place the Slide on the Stage
   • Open the stage clip and insert the slide so that the specimen is centered over the light source.
   • Use the stage adjustment knobs to position the specimen precisely.
4. Focus on the Specimen
   • While looking from the side of the stage, use the coarse focus knob to raise the stage until the objective lens is just above the slide.
   • Then look through the ocular lenses and slowly adjust the stage with the coarse focus knob until the image comes into view.
   • Use the fine focus knob to sharpen the image.
   • You may need to adjust the amount of light reaching the slide to get a clear view of your specimen. Too much light can wash out details; too little light can make the image dim. You can do this by adjusting the level of light coming from the light source as well as the iris diaphragm to control brightness and contrast.
5. Increase Magnification
   • Center the specimen in view before switching to a higher-power lens.
   • Rotate the nosepiece to click the next objective into place (you will rotate clockwise bring first the 10x and then 40x into position).
   • Use only the fine focus knob to adjust clarity — never the coarse focus knob on high power as this may cause the lens to collide with the slide. Remember, the microscope is parfocal, so only minor adjustments should be necessary.
6. Using the Oil Immersion Lens (100x) (if applicable)
   • Focus the specimen first using the scanning power/4x objective lens.
   • Add a drop of immersion oil directly on the cover slip.
   • Rotate the 100x objective lens into place so it touches the oil.
   • Use the fine focus knob to sharpen the image.
   • When finished, clean the lens and slide with lens paper only.
7. After Use
   • Return the objective to the 4x scanning lens.
   • Lower the stage, remove your slide, and turn off the light.
   • Unplug the microscope and wrap the cord and return the microscope to the proper location in the microscope cabinet.

Parts of the Dissecting Microscopes/Stereo Microscopes

• Ocular lenses (eyepieces): Our microscopes are binocular which means that there are two ocular lenses for you to look into. The eyepieces are connected by a moveable slider allowing you to move them closer together or farther apart to adjust to the distance between your eyes. Each has a magnification of 10x.
• Zoom knob: Adjusts magnification continuously as you rotate the knob. Our microscopes can zoom in for a maximum of 10x magnification.
• Focus knob: This knob is used to sharpen the image and bring the specimen into focus.
• Stage plate: This is the platform where you will place the specimen. Often, we will use petri dishes or a similar container to hold the specimens that we are observing with the dissecting microscopes. The platforms for our microscopes have a plate which can be removed and replaced. There is a clear plate and a black plate depending on what you want to observe and whether you want illumination from below.
• Upper light (incident light): Illuminates specimens from above.
• Lower light (transmitted light): Illuminates specimens from below.

Using the Dissecting Microscopes/Stereo Microscopes

1. Set Up the Microscope
   • Obtain a microscope from the microscope cabinet using care to place a hand under the base as well as the arm of the microscope to carefully carry the microscope to your workstation.
   • Turn on the appropriate light source: The upper light is often used for solid, opaque specimens while the lower light is typically used for transparent or thin specimens; both lights can be used together for best illumination. Note that there is one master power switch as well as two separate switches for each of the light sources. The master power switch must be turned on prior to turning on either of the light sources.
2. Place the Specimen on the Stage Plate
   • Position the specimen in the center of the stage plate.
3. Adjust Magnification and Focus
   • Look through the ocular lenses and use the zoom knob to select the desired magnification. Start at low magnification to locate features, then zoom in for closer inspection.
   • Use the focus knob to bring the image into clear view.
   • Adjust lighting as needed for the best contrast and detail by moving the dial for the light source(s) you are using.
4. After Use
   • Turn off the lights and unplug the microscope.
   • Remove any specimens and clean the stage.
   • Wrap the cord and return the microscope to the proper location in the microscope cabinet.