16
Sampling and
Digitization
After the optical phenomena have been discussed
in Part 1, Part 2 takes a closer look at how the digi-
tizing process and system-inherent sources of
noise limit the performance of the system .
As stated in Part 1, a confocal LSM scans the spec-
imen surface point by point. This means that an
image of the total specimen is not formed simulta-
neously, 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 fea-
ture to be resolved.
Confocal microscopy, especially in the fluores-
cence 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
fluorochromes, the transmittance of optical ele-
ments etc. (
see Details “Fluorescence”
). An addi-
tional influence factor is the energy loss connected
with the reduction of the pinhole diameter.
In the following passages, the influences of scan-
ning 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 consist-
ing of two self-luminous points spaced at 0.5 AU
(
see Details “Optical Coordinates”
). The diffraction
patterns generated of the two points are superim-
posed in the image space, with the maximum of
one pattern coinciding with the first minimum of
the other. The separate visibility of the points (re-
solution) depends on the existence of a dip
between the two maxima (see figure 12).
Part 2
337_Zeiss_Grundlagen_e 25.09.2003 16:16 Uhr Seite 19
