Biology Reference
In-Depth Information
8. The technology is working with a huge amount of data, and
therefore, it is very demanding of data storage capacity ( see
Note 29 ).
9. To use the outputs in other applications, the native “.vsi” data
format can be converted to the required format (e.g., “.jpg”
or “.tiff” fi les).
3.6 Specifi c
Considerations for
Arabidopsis Seedling
Slides
1. Place slides with seedlings of an individual M2 family into the
slide loader ( see Note 30 ).
2. For scanning of highly transparent objects such as are the
cleared Arabidopsis seedlings, it is necessary to set up particu-
lar parameters as follows: (1) completely close the aperture
stop and (2) set the “shade avoidance” and “white balance” in
the calibration settings ( see Note 31 ).
3. Initiate the reading of slides in the slide loader by selecting the
“batch scan” mode.
4. Set up the sample information and then set the options for
detailed scanning as follows: In the “more options” menu,
select acquisition to the highest sensitivity of sample detection,
“focusing sample only,” and check off the “auto adjustment of
scanning area” option. Specify the location at the storage
media where the image fi les will be saved and the fi le designa-
tions. Choose those slides that you want to scan and set the
scanning of slides with objective 10× (Fig. 2 ).
5. Start scan.
6. After the overview scan is fi nished, specify manually the whole
slide as the scanning area ( see Notes 26 and 32 ) and proceed
with the scanning ( see Note 33 ).
7. Every slide in the loader will be processed according to the
aforementioned settings. The average time when using these
settings is approximately 15-20 min per slide, and the fi nal fi le
size is approximately 300 MB.
Based upon our experience, we recommend manual image analysis
to identify mutants with changes in the expression profi le of the
gene of interest ( see Note 34 ).
3.7 Image Analysis
1. All loaded samples are by default saved in the native “.vsi”
format.
2. The “.vsi” format allows reliable viewing of the virtual slide
using your computer's mouse wheel to change the magnifi ca-
tion in a manner similarly to that of the Google Maps applica-
tion. A sample fi le of the entire virtual slide of 7 days old
ProCKI1:GUS M2 family seedlings scanned using a 10×
objective can be downloaded from http://www.ceitec.eu/
programs/genomics-and-proteomics-of-plant-systems/func-
tional-genomics-and-proteomics-of-plants/ . To view the
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