Micro-PIXE analyses of frozen-hydrated semi-thick biological sections

Cryo-micro-PIXE system and methodology of microanalysis of frozen-hydrated semi-thick biological sections is described. A commercially available cryotransfer system used in electron microscopy has been adapted for this purpose. The analyzed material was frozen by metal-mirror method and sections of 20-50 micron thickness were prepared. Micro-PIXE and simultaneous proton backscattering was performed using 3 MeV proton beam. Monitoring of water vapour composition during the proton bombardment showed good stability of the analyzed material. The results of repetitive analyses of standards prepared from gelatin-glycerol solution with added known concentrations of K, Ni, Cu, Zn were in good agreement with expected, calculated values. Mass losses and changes of elemental composition were monitored. Elemental maps obtained for frozen-hydrated semi-thick section of Ni hyperaccumulator Senecio coronatus showed excellent preservation of leaf morphology and the distribution of elements. Quantitative elemental mapping of frozen-hydrated specimens compared with subsequent analysis of the same areas after freeze-drying revealed similar distribution pattern in both cases. It is clear, however, that freeze-drying induces some distortion of cell morphology and specimen shrinkage.