Biology Reference
In-Depth Information
rate of 0.5 Gs/s (corresponding to 20,000-25,000 data points
per spectrum). Make sure there is enough space on the hard disk.
3. Perform mass calibration using a 1:1 mixture of PEG200 and
PEG600, diluted to 1:300 in TA30 solution (30 %, v/v, acetoni-
tril, 0.1 %, v/v, TFA) deposited next to your sample on the slide.
4. Adjust laser power and number of laser shots needed to a rela-
tive signal intensity of 25,000 by checking signal intensity from
various regions of the tissue section.
5. Create a fi le for MSI measurements using fl exImaging software
(Bruker Daltonics GmbH). Here, the optical image of the
sample (Subheading
4.1
,
step 6
) is needed to align sample
stage position and laser spot focus of the instrument. Designate
the measurement region and select desired spatial resolution.
For very small sections (such as young seed cross sections) use
15-20
m laser raster in order to get suffi cient representation
of the various tissues (
see
Note 7
). The autoXecute method
chosen defi nes i.e., number of laser shots and the fl exControl
method. Additionally, mark a small measurement region with
just matrix as blank of approximately 25-35 laser raster points.
6. Perform MSI measurement. Carry out the measurement of the
spots in random order to eliminate infl uence of measurement
order (This will result in longer acquisition time compared to
FAST modus. Necessity should be tested depending on tissue
properties and fi nal number of single spectra acquired in one
imaging run.)
μ
Methods are described for using fl exImaging software.
4.4 Data Analysis
for Small Molecule
MALDI-MSI
1. FlexImaging software is used for distribution analysis of
selected
m/z
signals. Not to include matrix derived signals,
overlay sum spectra of the tissue measurement region and of
the blank region (
see
Note 8
). Avoid selection of
m/z
values
that overlay completely with blank derived signals. It is possi-
ble to create a mass fi lter list with manually selected signals.
2. Using the mass fi lter list option multiple ion intensity maps can
be created. Colors for distinct mass signals to be displayed in the
multiple ion intensity maps should be chosen as the distribution
patterns of the individual single signals are then clearly observ-
able. In addition, individual ion intensity maps can be visualized
for representation of particular molecular ion abundances.
3. With the “Region of interest” (ROI) option different parts of
the measured sample can be chosen for comparison of sum
spectra according to these specifi c regions. For statistical analy-
sis the spectra lists of the ROI's can be exported for further
analysis using e.g., ClinProTools software (
see
Note 9
).
4. Targeted analysis for identifi cation of compounds.
