Automated Microscopy in Forward Genetic Screening of Arabidopsis - Plant Cell Morphogenesis: Methods and Protocols - page 63

drying. Before scanning stored samples, allow them to
acclimate to room temperature so as to avoid water condensa-
tion on slides and cover slips.
22. Bar code-labeled slides are saved after scanning using the bar
code information implemented in the resulting fi le. Any slides
without bar code are saved numerically in the default settings,
which also can be changed ( see Note 23 ). The system also
allows scanning the slide label area and saving this information
as an integral part of the virtual slide. This, however, increases
the fi le size.
23. Optionally, the following information can be added manually
for each slide to the synoptic fi le table: bar code, name, infor-
mation, specimen, species, tissue, preparation, staining, tray
number, slide number, and tray bar code.
24. Magnifi cation depends on screening type. For the screening of
seedlings 7 days old, a 10× objective is suffi cient ( see Fig. 2 and
sample data online).
25. Quality of the automatic scanning process depends greatly on
the setting of the scanning properties. In the acquisition mode,
sample detection sensitivity (high or low) during the scanning
process can be set. In the highest-sensitivity mode, even a low-
contrast specimen, but also most of the impurities on the slide,
is detected by the software. At the lowest-sensitivity setting,
on the other hand, you will be able to detect and automatically
focus upon only strongly contrasting objects. Finally, for
autonomous slide scanning, it is important to set the density
of the focusing map ( see Note 28 ).
26. For scanning of transparent samples, we recommend to adjust
the scan area manually to take in the whole slide. Scanning of
the entire slide ensures that all of the specimens will be scanned
at an acceptable quality, even if the automated recognition on
the slide overview fails to identify all specimens or their parts.
This can happen quite often, particularly in the case of trans-
parent and tiny Arabidopsis root.
27. EFI staining combines multiple images taken in different
planes of focus into a single, layered image. This ensures the
best image quality (by increasing the depth of focus) of the
specimen. However, using EFI dramatically increases both
scanning time and fi le size.
28. The focusing map is generated to avoid the need for focusing
the respective objective in each fi eld of view on the slide and
thereby to accelerate imaging. After the specimen is identifi ed in
the image overview, the microscope focuses upon the specimen
in a pattern, the density of which was set up as a scanning param-
eter ( see Subheading 3.5 , step 2 ). The position of focusing