Biology Reference
In-Depth Information
6. Perform MSI measurement. Carry out the measurement of the
spots in random order to eliminate infl uence of measurement
order.
Data analysis is performed in the same way as stated in
Subheading
4.4
.
6
Distribution Pattern of Small Molecules in Developing Barley Grains
Kernels of developing grains are composed of several different tissue
types undergoing massive changes during the fi rst 3 weeks after pol-
lination. The MALDI MSI approach is used to unravel distribution
pattern of compounds showing tissue and developmental specifi city.
Here, the advantages of this MS method are obvious as dissection
and micro-extraction procedures for these minute amounts of tis-
sues would require extreme efforts. Kernels of different time points,
representing major developmental stages are analyzed. In our analy-
ses several detected
m/z
values were found to be highly related to
distinct tissues. The multiple ion intensity map in Fig.
2a
demon-
strates accumulation of fi ve individual molecular ions in different
seed regions. As mentioned in Subheading
4.4
, statistical analysis is
performed using ClinProTools software. For this purpose regions
of interest (ROIs) were manually defi ned as indicated in Fig.
2b
and
subsequently used for statistical analysis. The PCA result for eight
ROIs and 131 individual
m/z
values shows high correlation of
molecular ions to distinct ROIs, displayed by defi ned groups
(Fig.
2c
). These results clearly illustrate the ability of the presented
MALDI MSI approach to signifi cantly assign distribution pattern
of molecular ions to defi ned plant tissues.
7
Notes
1. Regarding optical images of the sample, one has to record low
resolution images (<10,000 dpi) which provide a reasonable
overview of the sample and can serve as a reference image to
defi ne measurement areas. Most scanners provide reasonable
images, but in our hands an OpticLab H850 histological slide
scanner (Plustek Technology GmbH, Germany) proved very
useful. For actual histological evaluation, low resolution images
are typically not suffi cient. Although high-resolution images
from digital cameras and microscopes can be co-registered with
the MSI results, their fi le size often prevents effi cient use. Here,
microscope slides scanners such as the Pannoramic Desk (3D
Histech, Hungary) or ScanScope CS (Aperio, CA, USA) pro-
vide an elegant alternative, automatically scanning the slide at
several resolution levels in an approach similar to the one used
