Confocal Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 224

in Fig. 6.11b uses a linear CCD detector (as in the Lasertec microscope) and is
useful for metrological work as geometrical distortion is absent, and calibration
is determined by the dimensions of the CCD. In Fig. 6.11 c, line illumination is
combined with a slit (as in the former meridian system), and the confocal image
written on to a CCD detector (or directly observed by eye) by a coupled y 0 scan
[ 21 ]. The major advantage of this arrangement is that a complete line of information
can be recorded simultaneously, thus greatly increasing the optical throughput of the
system. Axial resolution and optical sectioning are somewhat degraded by the slit
geometry, especially for very thick samples, but this is overweighed by the ability to
image in the fluorescence mode at TV rates. In Fig. 6.11 d, line illumination is again
combined with a confocal slit, but this time the normal beam splitter is replaced by
a narrow strip mirror (Achrogate, by Zeiss) that reflects the illuminating light but
allows the fluorescent light from the sample to be detected [ 22 ].

6.4 Confocal Techniques

6.4.1 Basic Modes

Confocal microscopy is usually performed either in an epi-fluorescence mode
or a brightfield reflection mode. Reflection microscopy can be used for imaging
surface and multilayer structures, but when imaging thick biological specimens,
coherent noise can result in speckle artefacts. These coherent artefacts are avoided
in fluorescence microscopy. In addition to fluorescence and reflection imaging,
the confocal technique can be combined with any imaging mode of conventional
microscopy, including dark-field [ 23 ], polarization, Nomarski DIC [ 24 ] and inter-
ference microscopy [ 25 ]. In confocal transmission, a problem is encountered that
the light spot can move relative to the confocal pinhole as a result of the refractive
effects of a thick sample. This problem can be overcome using adaptive optics
techniques, but confocal transmission microscopy does not allow for 3-D imaging
in the same way as reflection microscopy, and the benefits of doing it are not
clear.

6.4.2 Spectroscopic and Nonlinear Methods

In principle, any form of spectroscopy can be combined with scanning to form an
image [ 26 ]. These include absorption spectroscopy, Raman spectroscopy, resonant
Raman spectroscopy, CARS, two-photon absorption spectroscopy, two-photon
fluorescence spectroscopy, photoelectron spectroscopy and photo-acoustic spec-
troscopy. In addition, various other nonlinear methods such as harmonic generation
and parametric conversion can be used as contrast mechanisms.