6
Two-Dimensional Images
For examining flat specimens such as cell culture
monolayers, it is usually sufficient to acquire one XY
image to obtain the desired information. The same
applies if the specimen is a three-dimensional tissue
section of which a single optical section is represen-
tative.
The thickness of the optical section (slice) and the
focal position are selected so that the structures of
interest are contained in the slice. The lateral reso-
lution of a 2D image is defined by the pixel size in
X and Y. The pixel size, in turn, varies with the
objective used, the number of pixels per scan
field, and the zoom factor. Pixels that are too large
degrade resolution, whereas pixels too small
require longer scanning times, bleach the speci-
men and generate superfluous data volumes. The
optimum pixel size for a given objective and a
given zoom factor can be set by selecting the
number of pixels with a mouse click.
The procedure for a two-dimensional image
1
Position and focus on the specimen in the
Vis
(ual) mode
2
Select the configuration to match the fluorochromes used
3
Define pixel resolution, scanning speed and, where required,
Average
Mode
4
Set the optical slice thickness by means of the pinhole diameter
5
Adapt the dynamic range to the specimen;
automatically via
Find,
or manually via
Gain
and
Offset
6
Adapt the scanning field to specimen substructures, using the
Crop
function
