Download Biology 3235: Resolution and magnification of a light microscopes

Biology 3235: Resolution and magnification of a light microscopes...
The total magnification afforded by a microscope is equal to the product of the
magnifications provided by the objective used and the oculars. For example, a 100× objective
used with 10× oculars gives a total magnification of 1000×: objects at the specimen plane will
appear 1000× larger.
While it would seem that objectives and oculars could be combined in microscopes to give
unlimited magnification, the "useful magnification" of a microscope is limited by the resolution of
the objective. Resolution refers to the ability of an optical instrument such as a microscope to
distinguish two closely-spaced objects. The theoretical resolution of any optical instrument,
including microscopes, telescopes, and cameras, is limited by the diffraction of light as it passes
through the lenses (or bounces off mirrors). The resolution of a microscope objective can be
expressed as a function of the wavelength of light (λ) and the numerical aperture (NA) of the
objective and condenser lenses:
R = 0.6 λ \ NA
As you can see from this formula, objectives with a larger numerical aperture give greater
resolving power (they can resolve smaller details). For example: a 10× objective with an NA of
0.25 will resolve details as small as 1.2 µm when using green light (λ = 500 nm). With a100×
objective (NA = 1.3), you could resolve details as small as 0.23 µm using green light. Note that
shorter wavelengths of light also provide for greater resolution; smaller details can be resolved
with blue light (λ = 480 nm) than with red light (λ = 650 nm).
The NA of dry lenses is limited by the refractive index of air, and the phenomenon of
internal reflection, to less than 1.0. By using oil between specimen and objective, numerical
apertures as high as 1.4 can be obtained. Thus the resolution of a light microscope equipped with
the best diffraction-limited oil immersion objectives is about 0.2 µm. Though in some cases
smaller details can be seen in light microscopes, they cannot be resolved (i.e.: two small organelles
less than 0.2 µm apart will be perceived as a single object).
The resolution of a microscope objective, in combination with the resolution of your eye
(a simple lens), also determines the maximum useful magnification of a microscope. The average
human eye under normal conditions can resolve objects as small as 100 µm. Dividing the
resolution of your eye by the resolution of the microscope objective gives the magnification
required to make all details resolved by the objective large enough to see with your eye. Greater
magnification is called "empty magnification," because it conveys no additional information. In
practice, maximum magnifications used are about two times the theoretical useful magnification,
to make details easier to see. Thus a 100× objective (NA 1.3) used with a 10× ocular gives a
1000× magnification, even though the theoretical useful magnification is only about 430×.
Developmental Biology Laboratory
University of Utah
Spring 2000