5
3
rd
dimension
In addition to the possibility to observe a single
plane (or slice) of a thick specimen in good con-
trast, optical sectioning allows a great number of
slices to be cut and recorded at different planes of
the specimen, with the specimen being moved
along the optical axis (Z) by controlled increments.
The result is a 3D data set, which provides infor-
mation about the spatial structure of the object.
The quality and accuracy of this information
depend on the thickness of the slice and on the
spacing between successive slices (optimum scan-
ning rate in Z direction = 0.5x the slice thickness).
By computation, various aspects of the object can
be generated from the 3D data set (3D reconstruc-
tion, sections of any spatial orientation, stereo
pairs etc.). Figure 4 shows a 3D reconstruction
computed from a 3D data set.
Time series
A field of growing importance is the investigation
of living specimens that show dynamic changes
even in the range of microseconds. Here, the
acquisition of time-resolved confocal image series
(known as time series) provides a possibility of
visualizing and quantifying the changes.
The following section (Part 1, page 6 ff) deals with
the purely optical conditions in a confocal LSM
and the influence of the pinhole on image forma-
tion. From this, ideal values for resolution and
optical slice thickness are derived.
Part 2, page 16 ff limits the idealized view, looking
at the digitizing process and the noise introduced
by the light as well as by the optoelectronic com-
ponents of the system.
The table on page 15 provides a summary of the
essential results of Part 1. A schematic overview of
the entire content and its practical relevance is
given on the poster inside this brochure.
Fig. 4 3D projection reconstructed from 108 optical slices of a
three-dimensional data set of epithelium cells of a lacrimal
gland. Actin filaments of myoepithelial cells marked with
BODIPY-FL phallacidin (green), cytoplasm and nuclei of aci-
nar cells with ethidium homodimer-1 (red).
Fig. 5 Gallery of a time series experiment with Kaede-trans-
fected cells. By repeated activation of the Kaede marker (green-
to-red color change) in a small cell region, the entire green flu-
orescence is converted step by step into the red fluorescence.
Introduction
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