Confocal Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 217

acts to control the spatial coherence of the overall imaging process, which can vary
between fully coherent (in which case field amplitudes add) and fully incoherent
(when intensities add). The aberrations of the condenser are not important, so that
the source and condenser together behave simply as a partially coherent effective
source. For this reason, in the widely used Kohler illumination, in which the
specimen plane is made conjugate with the field diaphragm rather than the light
source, imaging is in principle identical to critical illumination. If the numerical
aperture of the condenser is made small compared with that of the objective, imaging
becomes coherent. Coherent imaging exhibits fringing on edges in the image, and
phase information in the object (which results from optical thickness variations)
can be imaged as intensity changes if the system is defocused. Incoherent imaging,
on the other hand, is obtained if the numerical aperture of the condenser is made
large compared with that of the objective, and although performance depends on the
form of the specimen, generally incoherent imaging results in superior resolution.
It should be noted that in practice it is impossible to obtain truly incoherent
brightfield imaging when using a high-aperture objective and that in practice the
condenser aperture diaphragm is adjusted to give a compromise between resolution
and contrast in the image, resulting in partially coherent imaging. For the particular
case, commonly adopted in practice, where the numerical apertures of condenser
and objective are equal (so-called matched or full illumination), imaging, although
partially coherent, has similarities to incoherent imaging.
In Fig. 6.7 b, a scanning optical microscope is illustrated. A single point in the
specimen is illuminated by the focused and demagnified point source, whilst a
large-area detector, in conjunction with a collector lens, collects light from the
full field of view of the specimen. It will be observed that the ray paths shown
in Fig. 6.7 a and b are identical but reversed, and, because of the optical principles
of reciprocity and equivalence, the microscopes illustrated in Fig. 6.7 aandbhave
identical imaging properties. In the scanning optical microscope, Fig. 6.7 b, the
resolution of the system is primarily limited by the properties of the first lens,
called the objective or projector lens. The second lens, the collector, controls only
the degree of spatial coherence in the imaging process, and its aberrations are
unimportant. Because this optical arrangement is analogous to critical illumination,
it is called critical detection. An alternative arrangement, in which the detector
is placed in the back focal plane of the imaging lens, can be termed Kohler
detection: it behaves identically in principle but has the advantage that sensitivity
variations in the detector are not so important. Indeed, as the collector and detector
together act as an effective detector, the collector may in principle be dispensed
with altogether and the detector placed in that plane. Finally, in Fig. 6.7 c, we
combine the arrangements of Fig. 6.7 a and b to give a confocal scanning optical
microscope, in which a point source illuminates just a small region of the object,
and a confocal point detector detects light from this illuminated region. If the point
source and detector are scanned in unison, a two-dimensional image is generated.
However, this system behaves very differently from the previous ones. In fact, it
does not seem possible to devise a non-scanning system that behaves in the same