Biomedical Engineering Reference
In-Depth Information
Although microscopes used to be designed in the way presented Figure 8.11,
recent models are now corrected in a way to include a region within the microscope
where all the rays are parallel. The reason for using such geometry is fairly easy to
understand: what limits the performances of a microscope are the aberrations of the
optics. For good optics, these imperfections are limited but it is often necessary to
include optical components (filters, polarizers, etc.) in the optical path. In this case,
it becomes necessary to position these elements in a region of the microscope where
the rays are parallel. Modern research microscopes incorporate this feature and are
called infinity corrected systems.
Because of the diffraction, a single object appears as the convolution of the
object shape and a function called the Airy function. This means that, because of
the laws of far field optics, a point source will appear to have a finite size in the mi-
croscope. To be able to distinguish between two objects they have thus to be further
apart than the width of this function whose order of magnitude is the wavelength
of light. Thus, there is a separation criterion stating that the ultimate resolution of
an optical microscope is given by the classical Rayleigh formula:
d 1.22 (
=
λ
/ 2 N )
×
(8.4)
a
Where d is the smallest possible spacing between the two objects, λ is the wave-
length, and N a the numerical aperture of the microscope. If the objects are closer
than d, they appear as a single larger “blob.” To achieve a better resolution, high
numerical aperture objectives are needed (in particular oil immersion objectives) as
well as the use of blue wavelengths.
Still, any object regardless of its size can be observed by optical microscopy
provided that it emits enough photons. If it is too small, its observed lateral size has
nothing to do with the true one but if one is interested in its dynamic behavior or
in a more macroscopic measurement such as concentration, this is not a concern.
Fluorescence imaging of single molecules that is now routinely performed in many
laboratories is a good illustration of these possibilities. In the same line, tiny dis-
placements down to a few nanometers can be detected by optical interferometric
techniques [23].
Figure 8.11  Optical path of a microscope. The object to be observed is before the focal point of
the objective, close to it. Its image is formed at the focal point of the ocular, sending the final image
to infinity.
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