Confocal Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 225

6.4.3 Differential Phase Contrast

The non-confocal differential phase contrast (DPC) imaging mode is achieved by
using a detector split into two halves, in place of the PMT, the DPC image being gen-
erated by displaying the difference between the signals recorded from these semicir-
cular detectors [ 27 ]. This can be performed in either scanned-beam or scanned-stage
systems. Transmission-mode DPC has been used to see single-molecular thickness
variations in the sample [ 28 ]. DPC has been offered commercially by LaserSharp
and Olympus. The DPC image is effectively an image of the phase gradient,
modulated by the reflectivity or transmission of the object. A quadrant detector can
give the phase gradient in both x and ydirections. Further electronic processing of
the DPC image and a conventional one, generated by adding the signals from the
two detector halves, can be used to extract the phase gradient alone and then by
integration a pure phase image produced.

6.4.4 Optical Beam-Induced Current (OBIC)

In the optical beam-induced current (OBIC) technique, the focused laser spot is used
to generate electrical carriers in the specimen and the resultant current monitored. In
this way, the electrical properties of the sample can be imaged. It has been used with
semiconductor materials and devices, but there is the potential for other applications.

6.5 Imaging Performance of the Confocal Microscope

6.5.1 Resolution

For an ideally small confocal pinhole, the image intensity for a point object in a
reflection system (or a fluorescence system neglecting the Stokes shift) is given
by the square of that in a conventional system, as shown by the solid curve in
Fig. 6.12 . The image intensity exhibits a sharpening of the central peak of the Airy
disk, narrower by a factor of about 1.4, accompanied by very weak outer rings. The
curves are plotted against a normalized coordinate v , related to the true transverse
displacement r by v D krn sin ˛,wheren is the refractive index of the immersion
fluid, ˛ is the angular semi-aperture of the objective and k D 2=.
The axial image of a point object is also sharpened up by a true confocal system,
as shown in Fig. 6.13 . Here the curves are plotted against the normalized axial
coordinate u D 4kn z sin 2 .˛=2/,where z is the true axial distance. Also shown is
the axial image of a planar object, either in reflection (a) or in fluorescence (b). The
image of a plane in reflection is very sharp, as a result of coherent phase effects.