Page 230 - Fundamentals of Light Microscopy and Electronic Imaging

ADVANTAGES OF CLSM OVER WIDE-FIELD FLUORESCENCE SYSTEMS 213

than a focused point. The decreased transmission of photons through the pin-
hole is seen as a darkening on the TV monitor. The rejection of out-of-focal-
plane signals is easy to observe.
• If the coin is moved around on its support plate so that it is scanned by the
fixed laser beam, the pinhole screen and monitor show bright and dark sig-
nals that look like flickering, but an actual image is not observed. This rein-
forces the idea that the confocal signal is a continuously changing voltage,
not an image. In the scan head of an actual confocal system, the voltage sig-
nal is digitized at regular intervals into pixel values that are stored in a com-
puter in an image frame buffer board and displayed on a computer monitor.

ADVANTAGES OF CLSM
OVER WIDE-FIELD FLUORESCENCE SYSTEMS

The principal gain in imaging performance using CLSM is the ability to optically sec-
tion through fluorescent objects up to 10–50 m thick or to obtain high-contrast images
of surface topologies using confocal reflection optics. Contrast and definition are
improved, sometimes considerably, due to the reduction in background signal and a
greatly improved signal-to-noise ratio. With a stack of images representing different
focal planes spaced at regular intervals along the optic axis (the z-axis), the object can
be displayed in a variety of ways:

• Composite or projection view, where all of the information contained in a stack or
series of images taken at a series of focal planes along the z-axis is projected into a
single focused image. Consider a three-dimensional object such as a fluorescent
neuron with thin extended processes in a tissue section. In a single wide-field fluo-
rescence image, out-of-focus processes appear blurry and indistinct. A single con-
focal image shows the neuron extensions as fragmented streaks and dots, but a
composite view of all of the focal planes contained in a stack or z-series shows the
processes as continuous well-defined projections (Fig. 12-7).
• Three-dimensional views of an object which can be obtained from a z-stack of con-
focal images with the help of computer software for image display. Many confocal
software programs can display an object at different angular perspectives or rotate
the stack about the x-, y-, or z-axis, or a combination of axes, to view the cell in
three dimensions. This mode of viewing is valuable for examining complex three-
dimensional objects such as columnar epithelial cells and details in tissue sections
(Fig. 12-8).
• Transverse xz or yz cross-sectional views, which can be generated by most confo-
cal software programs. The object appears as if it had been cut transversely—that
is, in a plane oriented parallel to the optic axis.
• Five-dimensional views including information in x-, y-, and z-dimensions, in a
timed sequence, and in multiple colors. Such sequences can be displayed as a three-
dimensional multicolor movie in real time or time-lapse mode. Five-dimensional
viewing will become more convenient as computer processing speed and storage
capacity continue to improve.