Biology Reference
In-Depth Information
5. The optimal thickness has to be determined for each specimen.
In general, sections of 8-12
m thickness are ideal. From an
analytical perspective, an increased thickness of the sample rep-
resents a deviation from the optimal focus plane of the MALDI
laser and has negative effects on the focus of the resulting ion
beam, resulting in a loss of sensitivity. On MALDI-TOF instru-
ments in particular, an irregular depth profi le of the sample can
also induce electrical charging of the instrument source and
m/z
-shifts in the recorded spectra. In addition, thicker sections
have an increased risk to detach from the slide during or after
vacuum drying. However, a thickness below 15
μ
m is diffi cult
to obtain with non-embedded plant material, and 20-25
μ
m
sections are quite common. Tobacco root sections have to be
thicker than those of seeds to avoid disruption due to the large
intercellular spaces in the parenchymal cortex. Essentially, a
practical trade-off has to be made between morphological
integrity of the sample and the quality of the resulting mass
spectra. Thickness has a clear impact on the sensitivity of image
analysis. For tobacco roots, a slice thickness above 45
μ
m led
to a notable decrease in sensitivity. Same holds true for a slice
thickness above 40
μ
m in case of barley grains. For young (1-7
DAP) barley grains, 20
μ
m slices were effective, but as the
grain developed, the accumulating starch (associated with a
loss of the water content) forced the section thickness to be
increased to 30
μ
m. For some very delicate samples (such a
thin leaves or small roots) directly mounting the intact sample
onto an ITO slide is also an option. Double sided conductive
tape, such as Plano 16081-2 (Plano GmbH, Germany) can be
used to maintain conductivity.
6. The optical sensor of the ImagePrep monitors wetness and
thickness of matrix by voltage differences of the sensor. As
spray power during an application run can decrease, an auto-
matic spray power boost is implemented. Additionally, it is use-
ful to visually control spray application, especially if high
resolution images are desired as automatic spray power boost
can result in less uniform spray coverage and higher degree of
wetness.
7. The size of measurement region and chosen laser raster resolu-
tion defi nes the number of acquired single spectra. A very high
spectra number in combination with high number of laser
shots per raster spot can result in a signifi cant decrease in spec-
tra intensity due to pollution of the MALDI source. We have
generally chosen a maximum of spectra per run of ca. 15,000.
8. Small mass range analysis has the particular disadvantage of the
large number of background noise induced by fragmentation
and cluster formation of organic matrices during gas-phase
ionization. To elucidate this signals a small measurement
μ
