Three-Dimensional Images
To record the three-dimensional structure of a spe-
cimen, several two-dimensional optical sections are
made in different focal planes. The result is an XYZ
image stack, which can be visualized, processed and
analyzed.
The optical section is selected by shifting the posi-
tion of the focus in the specimen. This can be
effected by moving either the objective or the
specimen stage along the Z axis, according to the
microscope stand design. Whether the image
acquisition exhausts the resolving power given by
the objective’s numerical aperture depends on the
thickness of the optical slice and on the spacing of
two successive sections (the Z interval). According
to the Nyquist criterion, the optimum Z interval is
equal to half the optical slice thickness. If the pin-
hole diameter is selected to equal one Airy unit
(1AU), an optimum compromise between contrast
and intensity is achieved for the XY image. The
respective settings can be made by a mouse click
in the software.
If the sample to be examined is labeled with more
than one fluorochrome, it is necessary to adjust
the optical slice thicknesses of the various image
channels. The slice thickness is a function of the
numerical aperture of the objective, the wave-
length used, and the pinhole diameter. It differs
for channels detecting light of different wave-
lengths. In the systems of the Zeiss LSM 510 family,
every detector is equipped with a separate pin-
hole. This makes it easy to equalize the optical
slice thicknesses in the software – an important
condition for 3D colocalization analyses or for
reconstructing 3D images.
Once a 3D stack of images has been recorded, the
user has various presentation options. The data
may be displayed as a gallery of depth-coded
images or as orthogonal projections of the XY, XZ
and YZ planes. To create a 3D impression on a 2D
monitor, animations of different viewing angles
versus time, shadow projections, and surface ren-
dering techniques are possible.
8
The procedure for a three-dimensional image
1
Optimize the recording conditions for an XY image at the center
of the three-dimensional specimen (see box for 2D images)
2
Define the
Start
and
Stop
stack limits in the
Z Setting
menu
3
Define the optimum Z interval in the
Z Slice
menu
4
Acquire the Z stack
5
Display and analyze the stack in one of the
Gallery, Ortho
or
Cut
display modes
6
With multiple-labeled specimens, equalize the optical slice thicknesses in the
Z Slice
menu
