Focus & Stage Simulations

That you have to focus the microscope to clearly see the section on the slide is kind of obvious. When you focus on the section, you move the section into the focal plane of the lens you are using. What may be less obvious is that the focal plane is not a plane but rather a box. The thickness (or height) of the box depends on the microscope lens you are using. The thickness of the box decreases at increasing magnifications.

At low magnifications (x4 and x10) the thickness of the focal plane will typically be large enough to contain the entire section, i.e. you will be able to see all features of the section clearly. However,
when you use the x40 lens the focal plane is too shallow to contain the entire section, and you will only see part of the features of the section in focus.
If you are looking for specific cells it is sometimes worthwhile to browse the thickness of the section, i.e. to slightly focus up and down, instead of browsing the plane of the section, i.e. to move the stage.


Slide - Features Stain Magnification
testis, convoluted seminiferous tubule H&E x40
compact bone, Haversian system / osteon ground (unstained) x40
forebrain, mouse, cortex Giemsa x40
forebrain, rat, cortex Golgi x20
x100 - to oil or not to oil ...
... a little more about x100 lenses
The x100 lens is the lens with the largest magnification that you will find on light microscopes. That it has the largest magnification also means that it has the narrowest focal plane.
testis, convoluted seminiferous tubule, acrosomes PAS & iron haematoxylin x100
red bone marrow, rabbit, megakaryocyte H&E x100
spleen, reticular connective tissue reticulin x100
parathyroid gland, chief cells and oxyphilic cells H&E x100

Most x100 lenses require oil immersion, i.e. the space between the lens and the coverslip of the section is spanned by a drop of oil instead of air. Why that? Well, the refractive indices of air and glass are very different which will result in the scattering of light at the interface between the coverslip and the air and, again, at the interface between the air and the lens. The scattered light will obscure the finest detail visible in the section. The refractive index of the immersion oil is similar to that of glass, and light scattering will be minimal. If the oil is omitted x100 lenses resolve less detail than x40 lenses. The use a x100 oil lens requires all of the following:

Unfortunately, these conditions are rarely met in a teaching situation. Fortunately, there are only a few instances in which a x100 lens is really necessary to identify features of cells or tissues. The x40 lens will do the trick most of the time. The real power of the x100 lens is its very thin focal plane, which can provide a good idea of the 3-dimensional distribution of cells and tissue components in sections down to ~4 µm in thickness.