The Compound Microscope

The compound light microscope is by far the most utilized piece of equipment in the biologist’s tool chest.  While the origins are uncertain, by 1644 primitive versions of this microscope were used to visualize the interior of living tissue. Over the next several decades, the production of light microscope improved vastly and began to have a huge impact on our understanding of the natural world. Robert Hooke’s Micrographia, while not the first publication to illustrate this new knowledge, had a huge impact, in part due to his impressive illustrations showing immaculate detail of tiny insects, such as the flea and ant. Perhaps the biggest breakthrough resulting from early investigations using the microscope was the discovery of microorganisms first reported by Antonie van Leeuwenhoek in 1676.

A compound light microscope uses several mirrors to focus a light source on to the specimen. While these microscopes can be very simple, most biologists use specialized microscopes with features that allow them to control the resolution and contrast by manipulating the mirrors, light intensity and condenser aperture. 


Setting up the microscope

  1. Clean all of the microscope lenses using lens paper and lens cleaner.
  2. Open the arm of the slide holder and place the slide securely into it.
  3. Turn on the light. Determine where the light intensity control is. Turn it up and down looking through the ocular lenses.
  4. Locate the objective lenses on the revolving nosepiece. Point the lowest objective (usually 4x), toward the specimen. 4x is known as the scanning objective. It is the objective that we use to look at larger specimens under the microscope, or to locate regions of interest prior to switching to a higher objective. 


Visualizing the specimen

  1. With the scanning objective in place, center the specimen directly underneath it. At this point the stage should be all the way down.
  2. While looking through the ocular lenses use the coarse focus adjustment to raise the stage of the microscope until the specimen comes in and out of focus. Continue adjusting until the specimen is clear. If you need help, be sure to ask your instructor.
  3. Once your specimen is clearly in focus, locate the light intensity control and change the light intensity from low to high. 
  4. Turn the revolving nosepiece to the low power (10x) objective. Modern compound microscopes are confocal, meaning they maintain focus from one objective to another. At this point, use the coarse focus knob (and the fine focus knob) to focus the specimen.
  5. Once the specimen is in focus at low power, turn the revolving nosepiece setting the high power (40x) objective. Look at the distance between the lens of the objective and the microscope slide. USE ONLY THE FINE ADJUSTMENT KNOB TO FOCUS SPECIMENS AT HIGH AND OIL IMMERSION POWERS.
  6. Visualizing specimens properly requires specific lighting conditions.  While your specimen is in focus on high power, move the condenser aperture lever from left to right.
  7. Next you will look at your specimen with the oil immersion objective. As the name indicates you will immerse the objective lens in oil. The total magnification for the oil immersion objective is 1000x. At this magnification, light waves refract in such away that the resolution of the image is distorted. Immersion oil is a transparent liquid with a high refractive index, which allows the light to pass directly into the objective lens, enhancing the resolution. DO NOT USE THE COARSE ADJUSTMENT WITH THE OIL IMMERSION OBJECTIVE. ONLY USE THE FINE OBJECTIVE, BECAUSE YOU CAN BREAK THE LENS.

Principle of immersion microscopy. At high magnification power, light waves refract off the glass in the microscope slide and slip cover. Immersion oil has a high refractive index, minimizing this refraction allowing light to enter the objective in a straight line. This increases resolution of the specimen. 

Principle of immersion microscopy. At high magnification power, light waves refract off the glass in the microscope slide and slip cover. Immersion oil has a high refractive index, minimizing this refraction allowing light to enter the objective in a straight line. This increases resolution of the specimen.