Confocal Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 218

manner: we cannot generate a confocal image by placing a detector at any plane of a
non-scanning system. From the symmetry of Fig. 6.7 c, it is clear that the two lenses
play an equal part in the imaging process. We might expect that this would result
in an improvement in resolution, which is indeed the case. In fact, the confocal
system behaves as a coherent imaging system but with a sharper effective point
spread function than in a conventional coherent microscope. Although Fig. 6.7 is
drawn for a transmission geometry, in practice most confocal systems operate in
the reflection or epi-illumination mode, in which the same objective lens is used
both for illumination and detection. For a specimen placed in the focal plane, the
properties of confocal transmission and reflection systems are identical. However,
once the object is moved from the focal plane, the defocus properties behave
quite differently. Figure 6.7 could also equally well apply to fluorescence imaging.
However, in this case, because of the incoherent nature of fluorescence emissions
after excitation by coherent light, imaging is always incoherent. The Stokes shift,
the increase in wavelength of emitted light relative to the excitation, means that now
the scanning system of Fig. 6.7 b gives better resolution than the conventional system
of Fig. 6.7 a. The confocal system in principle gives superior resolution to either of
the other systems.

6.2 History of Confocal Microscopy

The confocal microscope arrangement is usually attributed to Minsky, who filed
a patent for the confocal microscope in 1957 [ 1 ]. However, in fact, the confocal
principle was described previously by others. In 1940, Goldman described a system
using line illumination and a confocal slit, rather than a pinhole, and presented
x- z cross-section images of human eyes [ 2 ]. Koana in 1943 and Naora in 1951
described a confocal method for reducing the strength of stray light in a photometry
system [ 3 , 4 ]. Laser illumination was incorporated into the confocal microscope
by Davidovits and Egger in 1969 [ 5 ]. In 1972, Slomba et al. described a confocal
fluorescence microscope that incorporated beam scanning [ 6 ]. The first commercial
confocal microscope was launched by Oxford Optoelectronics Ltd. in 1982. This
design was adopted by LaserSharp, which was subsequently taken over by BioRad.
The confocal microscope generates an image by illuminating the object point
by point with a spot of light. Petran described in 1968 a system that he termed the
tandem scanning microscope, in which the specimen is illuminated simultaneously
by many spots of light using an array of pinholes on a rotating disk (a Nipkow
disk) [ 7 ]. This arrangement allows for fast scanning and also for a confocal image
to be viewed in real time through an eyepiece. A problem with the Petran system
is that the Nipkow disk transmits only a small fraction of the light from the source.
Recently, this disadvantage has been overcome by focusing the light on to the disk
with a microlens array as in the Yokogawa system [ 8 ].