BIOL 227 (Fall 2023)

Lab 1: The compound light microscope

Learning outcomes

1. Describe the parts of the compound microscope (see hand out “How to use a
microscope”)

2. Describe the bending of light as it moves through the lenses (condenser, objective
and ocular lenses) and media (air, water and oil)

3. Explain how an increase in resolution is achieved as we move from scanning to

4. Practice the Kohler illumination step , oil immersion and dark-field illumination

A binocular microscope is equipped with two ocular lenses that are tilted toward the observer to
facilitate viewing. The ocular lenses also function to magnify the observed specimen. The typical
magnification of ocular lenses is 10X. The entire area (seen as a circle) observed through them is
called the field of view (FOV). Within the field of view of one of the ocular pieces, you will
notice a pointer. This pointer is useful when trying to show someone a specific feature on your
specimen. By moving the mechanical stage front to back and left to right, you can adjust the
position of the pointer. (Note: never attempt to remove any of the ocular pieces as they are very
expensive).

The objective lenses are fixed on a revolving nose piece. They include a scanning objective lens
(4X), a low-power objective lens (10X), a high-power objective lens (40X) and an oil immersion
lens (100X). Make sure to use the black ring on the nose piece when switching from one
objective lens to another. Written on each objective lens is the numerical aperture (N.A). This
value signals the resolving power of each lens. Thus, in addition to magnifying the image, the
objective lenses also improve the resolving power of the scope. Simply just magnifying the
image (called empty magnification) would produce a blurry image where two closely spaced
features on the slide will appear as one blur. As the resolving power increases the distance
between two points that can be resolved (ie be seen as clear and distinct) decreases. The Abbé
equation mathematically captures the resolving power of the microscope:

0.5
𝑑=
𝑛𝑠𝑖𝑛


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where  is the wavelength of light, n is the refractive index of the medium and sin is one half
of the angle of the cone of light emerging up from the slide and into the objective lens. The
numerical aperture (N.A) written on the objective lenses provides the value of the denominator
in the equation. In other words, N.A= 𝑛𝑠𝑖𝑛 .

Since both the ocular and objective lenses function to magnify the image, the total magnification
of specimens observed through a compound light microscope is the product of both lenses; this
also explains why we call it the “compound” microscope. In the images of a monocot leaf shown
here, the image on the left is observed on low-power while that on the right is observed under the
high-power objective lens; the total magnification that accompanies the images is therefore 100X
and 400X, respectively.

Total magnification: 100X Total magnification: 400X

The condenser lens is located immediately below the stage. The condenser lens does not magnify
the image but strongly impacts the resolution power of the microscope and the depth of field of
the specimen. The quality of the microscope is therefore largely determined by the craftmanship
of the condenser lens. Its function is to converge the light rays received from the circle of light
passing through the aperture iris diaphragm and bend them into a cone of light that strikes the
specimen. This change in orientation is because light gets refracted as it passes through a
medium of different density. In this case, the light went from air (n=1) to glass medium (n=1.52).

Inverted cone of light entering the objective lens

Cone of light striking specimen on slide

Circle of light crossing up and through the aperture iris diaphragm

(Image source: https://upload.wikimedia.org/wikipedia/commons/2/27/Condenser_diagram.svg)

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As the light passes through the specimen affixed on the glass slide, it is refracted again into a
now inverted cone as it re-enters the air. The Kohler illimitation protocol (see hand-out “How to
use the microscope) ensures that the rays of light within this second inverted cone enter directly
into the objective lens; a slight shift in the position of the condenser can greatly affect the
resolution power of the microscope. The Kohler illimitation protocol therefore lines up the
condenser lens with the objective lens. You should perform the Kohler illimitation protocol at
the beginning of each lab to get the best possible images from your microscope.

Note: To avoid scratching the precious objective lenses, always switch to the 4X objective lens before
removing a slide. Since the 4X has the greatest working distance, there is less of risk for the glass slide to
scratch the lens above it.

May not be copied or duplicated without express permission of author 3

Lab 1: The compound light microscope

1. Name the parts of the microscope.

(Source: Olympus CX41 Model Instruction Manual)

May not be copied or duplicated without express permission of author 4

2. Practice the Kohler illumination. In your own words, describe why this step is important.
You may include drawings.

3. View a slide under the 4X, 10X and 40X objective lenses (do not move the stage). Make a
drawing of your observations and include the total magnification for each. Next, for each
objective lens, measure the field of view (FOV) (in mm and µm using the micrometer slide
provided) and working distance (in mm only using a regular ruler).

4X 10X 40X

Tot. mag.: Tot. mag.: Tot. mag.:

a) Remove the micrometer slide and place back the specimen slide. Measure the size of the
specimen. Under which objective lens did you make your measurement and why?

b) In your own words, explain why the resolution improves as we move from the 4X to 40X
objective lens.

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4. Practice using dark field illumination. Observe a slide under bright-field and dark field
illumination. Draw your observations. In your own words, why does the dark-field stop
improve contrast.

5. Practice using oil immersion: Observe and draw your observations of a slide under the 40X
objective lens. Repeat with the same slide using the oil-immersion lens (for instructions, see
How to Use a Microscope document). Did you see an improvement in resolution? In your
own words, why does the resolution of the image increase when adding a drop of oil?

May not be copied or duplicated without express permission of author 6