Biology Reference
In-Depth Information
irreproducible. Some efforts have been made for solvent-free
approaches, for example via sublimation, but are currently not
applied for large scale analysis. Especially, for small molecule
MSI experiments carried out on little sample sizes a reproduc-
ible application technique with small droplet sizes as well as
high lateral resolution is essential.
3. As a simple rule, sample temperature is usually tied to the fat
content of the tissue in question. Low fat tissue is usually cut at
temperatures around −15 °C, whereas highly fatty samples may
require temperatures of −25 °C or lower. When cutting, observe
the behavior of the section. Sticky sections that cling to
the blade or the anti-roll bar usually indicate the temperature
should be lowered, while brittle and rupturing sections indicate
that the temperature is to cold. Plant tissues, with their usually
high water content, pose additional challenges when freezing
and may require the use of cryoprotectants. For protein analysis
of soybean cotyledons, we achieved good results using sucrose
solution (20 % w/v). Prior to freezing, the cotyledons were
placed in a syringe fi lled with sucrose solution and negative
pressure was applied, soaking the tissue with the cryoprotec-
tant. However, this method is not applicable for the analysis of
small molecule distributions as allocation of the molecular com-
pounds may likely be changed and signal intensity of analytes
will be suppressed by the very high sucrose level.
4. For the preparation of sections the use of polymeric resins
should be avoided, as these compounds will provide a strong
background and suppress signals of the tissue's analytes during
the subsequent MS imaging procedure. Most commonly used
embedding media, such as OCT or paraffi n contain polymers,
which can be washed from the samples using a combination of
water and/or alcohol washes. For example, dipping the sample
in warm, de-ionized water is usually suffi cient to wash out
OCT, and paraffi n can be removed by washing in xylenes for
10-15 min. While larger molecular weight peptides and pro-
teins usually do not suffer from extensive delocalization under
these washing conditions, more soluble analytes are usually
washed out from the tissue, so polymer-embedded tissue is
usually not suitable for the analysis of lipids or other small mol-
ecules. Alternative embedding media are methyl cellulose,
sucrose solution, gelatine, or simply water in case of protein
analysis. In contrast for very small, delicate samples, we have
had good success using poached egg white as an embedding
material for metabolite analysis. A small incision is made in the
egg white into which the sample is inserted and then both are
frozen in liquid nitrogen. The resulting, styrofoam-like prod-
uct can be easily cryo-sectioned and creates no noticeable
background signal.
