Biomedical Engineering Reference
In-Depth Information
Points of light from a specimen appear as Airy diffraction patterns at the
microscope intermediate image plane or back focal plane of the objective.
Each point of the specimen corresponds to a point in the Airy diffraction
pattern. The limit of resolution for an objective is its ability to distinguish
between two closely spaced Airy disks in the diffraction pattern. The
resolving power of a particular light microscope setup is mostly governed
by the ability of the microscope objective to capture more light - both
light diffracted by the specimen and undeviated light passing through the
specimen. This greater light capturing capability also allows the microscope
objective to better distinguish between two closely spaced Airy disks in the
diffraction pattern. Both inter-related conditions result in greater overall
resolving power.
Microscope objectives are the most important components of an optical
microscope because they are responsible for primary image formation and
determine the quality of images that the microscope is capable of produc-
ing. Objectives are also responsible for determining the magnifi cation of a
particular specimen and the resolution with which fi ne specimen detail can
be observed in the microscope. An optical property of the objective that is
important to the quality of the fi nal image produced by the microscope is
the numerical aperture (NA):
NA =
n
sin
α
[1.3]
where
n
= RI of medium between the objective front lens and specimen and
α
= one half angular aperture of the microscope objective.
This equation was also fi rst proposed by Ernst Abbe. The NA of a microscope
objective is a measure of its ability to gather light and resolve fi ne specimen
detail at a fi xed object distance. Usually the NA of an objective increases
with its magnifying power which results in the capture of more refracted
light rays and better resolution of fi ne detail. The resolution observed for a
particular light microscope setup can also be represented mathematically.
The resolution equation can be expressed as:
Resolution (
r
) = 0.61
λ
/NA (objective) = theoretical limit
[1.4]
Resolution (
r
) = 1.22
λ
/(NA (objective) + NA (condenser)) =
optical pathway
[1.5]
where
λ
= wavelength of the illuminating light and NA = numerical aperture.
To increase the resolving power of a particular microscope setup either
decrease the wavelength (
λ
) of the illuminating light or increase the total
NA of the complete optical pathway.
