CHAPTER 2 What Microscopes Do – Physically 25
The Useful Magnification
The highest magnification is not always the best for visualizing objects. The optimum
magnification depends on the structures you want to see. When selecting
a combination of eyepieces and objective lenses for an optimum magnification,
you need to take the numerical aperture (NA) of the objective into account. The
NA of an objective defines the objective’s resolution. A simple formula helps you
to calculate the useful magnification range:
Useful magnification to
500 1000
numerical aperture of
objective
In other words: with a numerical objective aperture of 1.4, the useful magnification
of the entire system is between 700× and 1400×. A magnification exceeding
this range results in a so called empty magnification. Empty magnification
occurs
when the image continues to be enlarged, but no finer details are resolved
and no additional information can be read out of the image. This is often the case
when higher magnification eyepieces are used.
Resolution in Terms of a Microscope
Resolution in microscopy is the ability to distinguish two objects from each other.
It is the minimum distance at which two distinct points of a specimen can still be
discriminated from each other. In a conventional light microscope, the smallest
resolvable distance between
neighboring structures is approximately 0.2 m. In
other words, the maximum resolution is 200 nm, which is due to the wavelength
( ) of light. This dependency was first observed by Ernst Abbe in 1873 and the
diffraction formula for lateral resolution thus named after him:
Abbe resolution /2NA
This value of 200 nm, however, is a quite theoretical one, which can only be
reached
under perfect conditions.
Beside the wavelength of light used to examine a specimen, other influencing factors
are also quite important. One main factor in determining resolution is the
numerical aperture (NA) of the objective as explained above. The higher the numerical
aperture, the better the resolution. But also the type of the specimen as