Symbiotic Fungi: Principles and Practice (Soil Biology)

14 Micro-PIXE Analysis for Localization and Quantification of Elements 229
Mesjasz-Przybyłowicz and Przybyłowicz 2002). Therefore, specimen cryo-fixation
is typically followed by specimen sectioning at low temperatures and finally by
freeze-drying (Frey et al. 2000; Schneider et al. 2002; Vogel-Mikusˇ et al. 2008b).
Even though cryo-sectioning is accepted as a routine preparation technique for
animal tissues, the number of studies that have used cryo-sections of fully differentiated
plant material for analytical purposes remains small. The primary reasons for
this are technical problems during freezing, due to the poor thermal conductivity of
plant tissues arising from the cellulose cell wall, a unique feature of plant cells, and
to the intercellular spaces filled with gas. Additionally, the alternating sequences of
different materials, e.g., the rigid cell walls, vacuolar ice crystals and gas-filled
intercellular spaces of the plant tissues, tend to result in sample crumbling during
either sectioning or freeze-drying, which makes it particularly difficult to obtain
cryo-sections with well-preserved morphology (Schneider et al. 2002).
The details of the more established cryo-preparation protocols for electron
microscopy cannot be easily adapted here. The sections required for micro-PIXE
analysis have to be much thicker (e.g., 60 mm) to obtain sufficient X-ray yield (Vogel-
Mikusˇ et al. 2008b), which in the case of micro-PIXE is acquired from the entire
depth of the specimen. Cryo-fixation must also be as rapid as possible to avoid the
formation of larger ice crystals, which could damage plant and fungal cells. In
addition, nuclear microprobe analyses are performed under vacuum conditions
(10 5 mbar or lower), and therefore the specimens must be dry and immobilized
in their preanalysis functional state (Vogel-Mikusˇ et al. 2007, 2008a, b).
14.2.3.1 Plant Material
Care should be taken regarding the physiological state of the material harvested for
micro-PIXE analysis, and therefore the use only of freshly harvested roots is
recommended, to avoid artificial element redistribution resulting from wilting and
senescence. The roots should be cleansed with tap and distilled water to remove all
of the soil particles. The secondary roots hosting mycorrhizal fungi should be
excised with stainless steel scissors to avoid sample contamination with metals.
14.2.3.2 Specimen Freezing
The freezing of the specimens should be done as rapidly as possible to reduce the
growth of ice crystals. Excised root fragments (cca. 0.5 cm long) are inserted into
stainless steel needles with polished tips (Schneider et al. 2002; Scheloske et al.
2004; Vogel-Mikusˇ et al. 2008b), which are carefully chosen according to the
root diameter, to ensure a tight hold of the root during sectioning (Fig. 14.1).
Alternatively, the excised root fragments can be transferred into aluminium foil
beds (0.5 0.5 0.5 cm) that are filled with tissue-freezing medium. Afterwards
the specimens are dipped into propane cooled with liquid nitrogen, which provides
a superior cryogen when compared to liquid nitrogen, due to its higher cooling rate.
A specially designed thermo-block is used for this (Fig. 14.1), which should be