Zeiss LSM 880 Operating Manual page 668

Sampling and
Digitization
After the optical phenomena have been discussed in Part
1, Part 2 takes a closer look at how the digitizing process
and system-inherent sources of noise limit the performance
of the system .
As stated in Part 1, a confocal LSM scans the specimen
surface point by point. This means that an image of the
total specimen is not formed simultaneously, with all points
imaged in parallel (as, for example, in a CCD camera), but
consecutively as a series of point images. The resolution
obtainable depends on the number of points probed in a
feature to be resolved.
Confocal microscopy, especially in the fluorescence mode, is
affected by noise of light. In many applications, the number
of light quanta (photons) contributing to image formation is
extremely small. This is due to the efficiency of the system
as a whole and the influencing factors involved, such as
quantum yield, bleaching and saturation of fluoro chromes,
the transmittance of optical elements etc. (see Details "Fluo-
rescence"). An additional factor of influence is the energy
loss connected with the reduction of the pinhole diameter.
In the following passages, the influences of scanning and
noise on resolution are illustrated by practical examples and
with the help of a two-point object. This is meant to be an
object consisting of two self-luminous points spaced at 0.5
AU (see Details "Optical Coordinates"). The diffraction pat-
terns generated of the two points are superimposed in the
image space, with the maximum of one pattern coinciding
with the first minimum of the other. The separate visibility
of the points (resolution) depends on the existence of a dip
between the two maxima (see figure 13, page 20).
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