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Micro-PIXE analysis of biological tissue in frozen hydrated state
Vavpetič, Primož (Author), Pelicon, Primož (Mentor) More about this mentor... This link opens in a new window

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Abstract
Since 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.

Language:English
Keywords:micro-PIXE, frozen hydrated specimen, focused ion beam, ion source, ion accelerator, biology, elemental analysis, elemental mapping
Work type:Doctoral dissertation (mb31)
Tipology:2.08 - Doctoral Dissertation
Organization:FMF - Faculty of Mathematics and Physics
Year:2018
COBISS.SI-ID:3261796 This link opens in a new window
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Downloads:332
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Secondary language

Language:Slovenian
Title:Analiza bioloških tkiv z metodo mikro-PIXE v zamrznjenem hidriranem stanju
Abstract:
Določanje porazdelitve elementov v bioloških sistemih na nivoju tkiv in posameznih celic z elementno občutljivostjo na nivoju ppm je vse večjega pomena v raziskavah kompleksnih procesov v bioloških sistemih. Določanje elementnih porazdelitev in koncentracij v različnih morfoloških strukturah določenega tkiva je ključnega pomena za razumevanje mehanizmov, ki so vključeni v regulacijo, razporeditev, absorpcijo, transport, kopičenje, funkcionalnost in dosegljivost elementov v bioloških tkivih. Glavni cilj mojega doktorskega dela je nadgraditev raziskovalne linije na mikrožarku za analizo zamrznjenih tkiv z metodo mikro-PIXE ter zmanjšanje premera protonskega žarka pod en mikrometer, ki nam bo omogočil vpogled v biološke strukture na podceličnem nivoju. Skonstruirali in sestavili smo kriostat s sistemom za hitro vstavljanje zamrznjenih vzorcev v vakumski sistem merilne komore. Kriostat, ki je hlajen s tekočim dušikom, nam med meritvijo omogoča vzdrževanje dovolj nizke temperature med meritvijo. Skonstruirali smo nosilec vzorcev, ki omogoča hitro vstavljanje zamrznjenih vzorcev in hkrati služi kot dovolj velik toplotni ponor, da preprečuje segrevanje vzorcev med vnosom in meritvijo. Vzorci so vstavljeni med dve tanki polimerni membrani debeline 100 nm. Med nosilcem vzorcev in membranama z vzorcem smo dosegli dovolj veliko toplotno prevodnost, ki ohranja vzorce v zamrznjenem stanju. Med nosilcem vzorcev in kriostatom pa smo vzpostavili dovolj velik toplotni stik, ki omogoča zadovoljivo odvajanje toplote z merilne mize. Nova in nadgrajena zasnova sprejemne vilice na roki goniometra lahko zdaj sprejme tako stari nosilec vzorcev kot tudi nov nosilec zamrznjenih vzorcev brez bistvenih razlik med njima in z istim transportnim postopkom za hitro vstavljanje vzorcev v merilno komoro. To znatno skrajša čas zamenjave vzorcev v merilni komori, ker ni potrebno evakuirati celotne postaje z mikrožarkom za izmenjavo vzorcev, kot pred nadgradnjo merilne postaje. Ker je potrebno zamrznjene hidrirane vzorce med merjenjem in med ekspozicijo v visokem vakumu hraniti zamrznjene in nedotaknjene, je bila potrebna popolnoma nova oblika nosilca za zamrznjene vzorce. Merilna miza, tj. novi nosilec zamrznjenih vzorcev, mora zadostiti večim pogojem, da bi vzorci ostali nedotaknjeni. Prvič, nosilec vzorcev mora zagotavljati dober toplotni stik med novo ročico goniometra s kriostatom in ga je treba narediti na način, da ga je mogoče prenesti v merilno komoro z novim transportnim sistemom. Nosilec zamrznjenih vzorcev mora biti dovolj velik, da lahko zagotovi zadosten toplotni rezervoar, da se vzorci ne bodo preveč segreli, če se z njimi rokuje približno eno minuto. Nosilec zamrznjenih vzorcev naj bi po možnosti vseboval več kot le eno ležišče za merilni vzorec, poleg tega pa mora biti v primeru nepričakovanega defokusiranja žarka tudi omogočeno vključevanje nekaterih orodij za fokusiranje in za preverjanje profila žarka. Znotraj mojega raziskovalnega dela smo pred kratkim v laboratoriju na pospeševalnik vgradili nov tip ionskega izvora, ki ima za faktor 15 boljšo svetlost od izvora Duoplasmatron, s katerim sem do sedaj izvedel vse meritve mikro-PIXE. Tako nam bo nov ionski izvor predvidoma omogočil zmanjšanje premera protonskega žarka pod en mikrometer in s tem omogočil raziskave bioloških tkiv na podceličnem nivoju, kar je tudi eden izmed ciljev mojega doktorskega dela. Pridobljeni mikro-PIXE elementni zemljevidi, pripravljeni kot liofilizirani v primerjavi z zamrznjenimi hidriranimi, kažejo pomembne razlike v elementarni porazdelitvi že na ravni tkiva. To je lahko posledica spremembe morfologije, zlasti tkivnih oddelkov, ki jih povzroča odstranitev vode med postopkom liofilizacije. Pridobljeni elementarni zemljevidi Daphnia magnae (slika 21, 22), pripravljeni le za zamrznjeno hidrirano mikro-PIXE analizo, kažejo, da je naša sposobnost merjenja biološkega vzorca večinoma sestavljena iz vode, kjer ni mogoče uporabiti liofilizacijske tehnike za pripravo vzorca brez večjih naporov, mogoča.

Keywords:mikro-PIXE, zamrznjen hidriran vzorec, fokusiran ionski žarek, ionski pospeševalnik, biologija, elementna analiza, elementno kartiranje

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