Imaging mass spectroscopy MeV – SIMS with continuous primary beamJenčič, Boštjan (Avtor) Pelicon, Primož (Mentor) Mass spectroscopyMeV-SIMSIon beam methodsMolecular imagingSecondary ion mass spectroscopy with swift primary ions, known under acronym MeV-SIMS, is a novel technique in imaging mass spectroscopy for detecting the distribution of heavy biomolecules in biological tissues. After its beginning in 2008 at Kyoto University, MeV–SIMS was launched in 2011 at Jožef Stefan Institute with the in-house construction of linear Time–Of–Flight (TOF) mass spectrometer and its installation at the high energy focused ion beam. In its initial configuration, the MeV-SIMS setup at JSI relied on pulsing of the primary ion beam. Most commonly used primary ion beam consisted of 35Cl6+ ions with the energy of 5.8 MeV. The characteristics of MeV-SIMS in this configuration were exploited in series of measurements on reference materials and biological tissue samples. Several case studies in pulsed beam configuration performed within this thesis indicate promising sensitivity for biomolecules in the range from 150 to 1500 Da, and their two-dimensional distributions were measured by lateral resolution in the order of 10 micrometres. The applicability of this configuration is strongly constrained by mediocre mass and lateral resolution. In the imaging mass spectroscopy, high mass resolution enables the recognition of mass peaks and their attribution to distinct parent molecules in the sample. High lateral resolution dominantly determines the applicability in biomedical research, as the molecular distributions of molecules on cellular and subcellular level are of particular interest. The main goal of my doctoral thesis was to improve the characteristics of the existing MeV–SIMS setup at JSI, and to demonstrate its applicability in biochemistry research. In order to do that, I concentrated on the instrumental approaches that would overcome the existing limitations in the lateral and mass resolution. Attention was first given to alternative approach, which enables molecular imaging without focusing and raster scanning the primary ion beam. Stigmatic imaging of secondary ions from the sample to a fast position sensitive detector TimePix by electrostatic lens enables imaging analysis with broad beam. Such approach is of high interest for MeV-SIMS community, as swift ions are difficult to focus down to micrometre dimensions due to their high magnetic rigidity. Best primary beams for MeV-SIMS available at tandem accelerators feature magnetic rigidities that exceed the capabilities of standard microprobe optics based on magnetic quadrupoles. We studied experimentally the properties of point–to–point transmission of secondary ions from a specific coordinate on the sample to the corresponding location at the TimePix detector by the einzel lens. We demonstrated the lateral resolution of 37 micrometres, which is not drastically inferior to the lateral resolution of MeV-SIMS with a pulsed primary ion beam. During the MeV–SIMS with a pulsed primary ion beam, primary ion beam is transmitted to the target within short time intervals of approx. 10 ns with a frequency of 10 kHz. Considering the primary ion beam current to be approx. 10 pA, such short pulses are often empty. Even if this is the case, the Time–of–Flight sequence needs to be carried out, which results in many blank waiting periods, and consequentially, in a time inefficient measurements. In order to compensate for this shortcoming, we must strongly increase the primary ion current by extending the size of beam defining slits at the microprobe. This results in mediocre lateral resolution in the range of 15 micrometres. Since the primary beam pulsing defines the start signal for the TOF cycle, the targeting challenge was to provide alternative source of the “start” signal. Within the thesis, we experimented with various alternative modes of triggering the TOF measurements. Triggering upon the detection of secondary electrons resulted in bad time resolution. The installation of the continuous electron multiplier (CEM) detector behind the sample resulted in well defined “start” upon the arrival of individual primary ions. In this way, the TOF cycle is triggered deterministically upon each individual arrival of the primary ion. It turned out, that a primary ion beam flux of 5000 ions per second is needed, corresponding to the intensity reduction of the primary ion beam by three orders of magnitude in comparison with the pulsed mode. Corresponding reduction of the beam definition aperture sozes at the microprobe forming system brings the beam size down to the sub-micrometre regime and the associated lateral resolution well suited for cellular and sub-cellular imaging of biological tissue. In addition, the associated excellent time definition of the “start” in the TOF cycle resulted in significant improvement in the mass resolution. The occurrence of background induced by stochastic arrivals of additional primary ions during the TOF cycle was supressed by beam blanking within the duration of the TOF cycle. In this way, we managed to record mass spectra with low level of instrumental background. The limited range of swift ions in biological tissue represents a serious problem for the new MeV-SIMS approach. In order to overcome this constraint, we redesign the sample substrates and the tissue preparation protocols. The appropriate substrate for MeV-SIMS with continuous primary beam consists of 100 nanometre thick pioloform foil, double-side coated by graphite to ensure its electric conductivity, spanned over the aperture of an aluminium holder. Colleagues from Biotechnical faculty at University of Ljubljana prepared thin biological tissue slices with thickness well below 4 micrometres and deposit them on the substrate. The MeV-SIMS analysis of the tissue samples with continuous primary ion beam demonstrated the suitability of this technique for molecular imaging at the level of individual cells. Since the primary goal of MeV-SIMS method is to detect non-fragmented biomolecules with masses of over 1 kDa, we estimated a high mass limit of detection with the existing MeV–SIMS setup. The TOF spectrometer relies on the detection of secondary ions by micro-channel plate detector positioned at the end of the drift tube. With heavier molecules, the velocities become too low for the efficient detection. The efficiency of detection was measured for various acceleration voltages and ion masses. The estimated detection efficiency for secondary ion with mass of 3 kDa falls well below 1 percent. This critical feature can be corrected with a detector, allowing strong post-acceleration of secondary ions before the strike at the sensitive surface of the detector. Alternative costly solution is to incorporate orbitrap spectrometer for MeV-SIMS, which detects radial oscillation of secondary ions, trapped in spindle-shaped electrostatic field.20182018-09-15 07:45:01Doktorsko delo/naloga103225sl