Biomedical Engineering Reference
In-Depth Information
The microscope can be operated using either a transmitted or refl ected
illuminating light path. The choice of which illuminating light path to use is
determined by the specimen being either transparent or opaque. Examples
of transparent specimens are cell suspensions and electrospun nano-fi ber
scaffolds while an opaque specimen would be the surface of an implantable
dental ceramic.
In transmitted light microscopy the light follows a well-defi ned optical
pathway within the various lens containing components of the modern
research microscope. The light travels from the illuminating light source,
usually a tungsten halogen bulb, passing through both collector and fi eld
lenses before being refl ected into the substage condenser by the fi eld dia-
phragm assembly and converged onto the specimen. After passing through
the specimen, image forming light rays are captured by the microscope
objective and passed either into the eyepieces or directed by a beam splitter
into one of several camera ports.
The optical pathway in transmitted light microscopy is shown in Fig. 1.3,
together with the two co-incident imaging and illuminating light paths.. The
condition shown in Fig. 1.3 is a visual example of Köhler illumination which
was fi rst introduced in 1893 by August Köhler of the Carl Zeiss corporation as
a method of providing optimum illumination of the specimen.
1
Illumination of
the specimen is the most important variable in achieving high-quality images
in microscopy and critical photomicrography. In Köhler illumination an image
of the illuminating lamp fi lament is critically focused sequentially at the fi eld
diaphragm, the substage condenser and the back focal plane of the objective.
Köhler illumination is used in both refl ected light and transmitted light micros-
copy. The details of setting up Köhler illumination in a bright fi eld microscope
is beyond the scope of the technical overview presented in this chapter - fur-
ther information can be obtained on the Web (online tutorial on Kohler illumi-
nation produced by the Light Microscopy Core Facility, Duke University and
Duke University Medical Center: http://microscopy.duke.edu/kohler.html).
1.3.2 Optimizing image resolution and contrast
The resolution and contrast of images obtained using light microscopy tech-
niques can be optimized by manipulating the various parameters of the
optical components of the microscope such as the numerical aperture (NA)
of the microscope objective and increasing the phase shifts or optical path
differences of the light waves that are diffracted by the specimen and pass
through the microscope's various lens assemblies.
Ernst Abbe's diffraction model of image formation
Microscope specimens can be considered as complex diffraction grat-
ings with details and openings of various sizes. Due to diffraction both
