Micro-PIXE analysis of biological tissue in frozen hydrated stateVavpetič, Primož (Avtor) Pelicon, Primož (Mentor) micro-PIXEfrozen hydrated specimenfocused ion beamion sourceion acceleratorbiologyelemental analysiselemental mappingSince mechanical damage, morphological changes and chemical oxidation during drying of biological samples still represent a major obstacle to keep the sample situation as in-vivo, technology of frozen hydrated tissue is expanding to research with nuclear microprobes. Slices of biological tissue after cutting are deposited on a dedicated support and under proper handling, the tissue sample remains in deep-frozen state and is reminiscent of amorphous glass. The main aim of my dissertation research was an upgrade of the high energy focused ion beam facility to be able to analyze frozen hydrated tissue with the micro-PIXE method. In parallel, we aimed to reduce the diameter of the proton beam down to sub-micrometer dimension, which would enable an insight into the biological tissue at the cellular and sub-cellular level. Cryostat, which is cooled with liquid nitrogen, allows us to hold samples at a sufficiently low temperature during the measurement. The cryostat is holding sufficient amount of liquid nitrogen and it lasts for 16 hours at least before all of the liquid nitrogen is evaporated. To best preserve the goniometer arm movement inside the chamber and to minimize any additional repulsion force for the goniometer stepper motors, the cold cryostat nozzle inside the chamber is connected to the goniometer arm where the cryo sample holder connects onto it by the accepting fork with a flexible braided pure copper cable acting as a heat-pipe. Constructed is the new measurement table i.e. cryo sample holder for the vacuum goniometer arm that allows quick insertion of frozen samples and provides an efficient heat sink to maintain the samples at low temperature inside the measuring chamber. Samples are inserted between two thin pioloform membranes. The cryo sample holder is made of aluminium metal, that was CNC machined to the highest degree of accuracy. The sample holder sandwiches are inserted into specially designed beds from behind and are then pressed into them with the special screw with the aluminium gasket in between. This ensures sample holder sandwiches not to be damaged in any way and also ensures good thermal contact between the main cryo sample holder housing and the sample sandwiches. The cryo sample holder can house four such holder sandwiches that are to be examined by the nuclear microprobe. Between the cryo sample holder and the cryostat we achieved sufficient thermal contact through the accepting fork, which ensures satisfactory heat dissipation from the cryo sample holder. Within my research work in the laboratory a new type of ion source on the accelerator was installed, which has brightness higher by a factor of 15 than the duoplasmatron ion source, with which I conducted all the measurements with micro-PIXE method so far. The increased beam brightness of the new ion source enabled us the reduction in proton beam diameter well below one micrometer and thus significantly improved the proton microprobe resolution. Additionally, the installation of the multicusp ion source strongly enhanced the accelerator up-time since H and He beams are produced by independent ion sources. With the addition of the new multicusp ion source, the microprobe beam line has been upgraded with an in-house built water-cooled motorized object slit, able to intercept of up to 1 kW of beam power. The comparison of the resulting elemental distributions measured at the biological tissue prepared as frozen-hydrated or freeze-dried revealed significant differences in elemental distribution of particular elements at the cellular level due to the morphology alteration in particular tissue compartments induced either by water removal in the lyophillization process or by unsatisfactory preparation of samples for cutting and mounting procedure before freeze-drying. The latest measurements on the Daphnia magna at the JSI nuclear microprobe introduced the ability to measure any kind of biological samples in frozen-hydrated state.20182018-10-27 07:45:09Doktorsko delo/naloga105121sl