Page 106 - Fundamentals of Light Microscopy and Electronic Imaging

SPATIAL RESOLUTION 89

fication, if the angular aperture of a microscope is increased, as occurs when opening
the condenser diaphragm or when changing the objective for one with the same magni-
fication but a higher NA, the diffraction spots in the image grow smaller and the image
is better resolved (Fig. 6-4). Thus, larger aperture angles allow diffracted rays to be
included in the objective, permitting resolution of specimen detail that otherwise might
not be resolved (Fig. 6-5).
The optical limit of spatial resolution is important for interpreting microscope
images. Irregularities in the shapes of particles greater than the limiting size (0.52 m
diameter in the example cited previously) just begin to be resolved, but particles smaller
than this limit appear as circular diffraction disks, and, regardless of their true sizes and
shapes, always have the same apparent diameter of 0.52 m. (The apparent variability
in the sizes of subresolution particles is due to variations in their intensities, not to vari-
ability in the size of their diffraction spots.) Thus, whereas minute organelles and fila-
ments such as microtubules can be detected in the light microscope, their apparent
diameter (for the lens given previously) is always 0.52 m, and their true diameters are
not resolved. It should therefore be apparent that two minute objects whose center-to-
center distance is less than 0.26 m cannot be resolved, but that two objects with phys-
ical radii smaller than this size can easily be resolved from each other if they are farther
apart than 0.26 m.
It must be remembered that adjusting the condenser aperture directly affects spa-
tial resolution in the microscope. Since a large aperture angle is required for maximum
resolution, the front aperture of the condenser must be fully illuminated. Stopping down

b′
a′
Back aperture stop
of objective lens

Objective
θ
Specimen

Condenser
a
b
Condenser
diaphragm

Figure 6-4
Role of the condenser diaphragm in determining the effective numerical aperture. Closing
the front aperture diaphragm of the condenser from position b to a limits the angle of the
illumination cone reaching the objective, and thus the effective numerical aperture. Notice
that the back aperture of the objective is no longer filled at the reduced setting.