In the last decade, there has been a growing interest in the elemental and molecular distribution on the tissue surface. These species are localized to a specific region of the tissue and play an important role in biochemical processes. Therefore, it is important to investigate the spatial distribution, structure and function of these biological species [1]. The current surface characterization techniques provide unique but limited information on lateraly-resolved elemental and molecular distributions. In order to overcome these limitations, we are using a dual mode imaging technique by combining micro-PIXE [2] and MeV SIMS [3,4] capable of providing us with the elemental and molecular distribution on the same tissue samples.

The MeV SIMS measurements are performed by a 5.8 MeV ³⁵Cl⁶⁺ primary ion beam focused to a dimension of 20 µm x 20 µm. The acquired molecular maps are then correlated with the sequentially measured elemental maps by micro-PIXE, measured at the matched sample region. Micro-PIXE maps are acquired by a 3 MeV proton beam and the current lateral resolution limit of 700 nm x 700 nm. The current status of the dual imaging technique as well as the tissue sample preparation protocols will be presented at various plant case studies, with an emphasis on the leaves of Al-treated tea plants (Camellia sinensis). The correlated elemental and molecular distributions on other tissue types, including rodent brain and cocaine distribution in human hair, will also be presented.

[1] Heeren, R. M. A., McDonnell L.A., Amstalden E., Luxembourg S.L., Altelaar A.F.M,. Piersma S.R Appl. Surf. Sci. 252, 2006,6827–6835.

[2] P. Pongrac et al., Journal of the Royal Society interface 10 (2013) no 84

[3] Y. Nakata, Y. Honda, S. Ninomiya, T. Seki, T. Aoki, J. Matsuo, Applied Surface Science 255 (2008) 1591-1594

[4] L. Jeromel, N. Ogrinc, Z. Siketić, P. Vavpetič, Z. Rupnik and P. Pelicon, Nuclear Instruments and Methods B, 332 (2014), 22-27

We present results of a new TOF-SIMS experiment designed to investigate electronic sputtering phenomena induced by swift heavy ions (SHI). This experiment has the unique capability to study not only emitted ions from the sample surface but their neutral counter parts as well. For this purpose the experiment is equipped with a F₂-laser to post-ionize the emitted neutrals and make them accessible to analysis with a TOF-spectrometer. The Laser is emitting light pulses at 157 nm wavelength (corresponding to a photon energy of 7.9 eV) which are used for single photon ionization of the emitted neutral atoms, molecules or clusters. The instrument is installed at the M1-Branch of the UNILAC beam-line at the GSI (Gesellschaft fuer Schwerionenforschung) at Darmstadt, Germany. The accelerator is able to provide ion pulses in the range from 3.4 MeV/u up to 13.6 MeV/u for all stable isotopes of the periodic system of elements. Besides the ion beam of the accelerator the instrument is equipped with a 5-keV Ar­⁺-Ion gun to perform comparison measurements in the nuclear sputtering regime and for alignment purposes.

In 2014 the instrument has been tested and special protocols for the use of the accelerator beams have been developed. The accelerator provides pulses of a length of 1-5 ms and a repetition rate of 1 to 50 Hz, which is very unusual for a SIMS/SNMS experiment. This fact leads to a completely different timing scheme especially for an effective use of valuable beam time. For this the extraction voltage is pulsed several times during a single accelerator pulse and during the breaks blank spectra, residual gas spectra and keV-SIMS/SNMS spectra are collected for an optimized use of the beam time and to ensure identical experimental conditions.

We present results collected during the beam time in 2014 with a special focus of SIMS and SNMS experiments on organic molecules.

Secondary Ion Mass Spectrometry induced by MeV heavy ions (MeV-SIMS) has emerged in recent years as a promising analytical technique for submicron mapping of molecular concentrations at the surface of organic and insulating samples. The numerous applications of this non-marking technique in art, archaeometry, forensics, or in the analysis of biological samples, as well as the possibility to extract a focused beam in ambient pressure, motivate interest.

The promise of the technique was also recognised by the International Atomic Energy Agency (IAEA), which has recently initiated a Coordinated Research Project (CRP No. F11019) to support the development of molecular concentration mapping techniques using MeV focused ion beams. The aim is to provide robust theoretical and practical foundations and credibility to MeV-SIMS.

Within this CRP, a first round robin exercise has been carried out in order to outline best practice methods for sample handling and measurement protocol. Valuable insight in some fundamental parameters associated with mass spectrometry imaging is investigated. Samples of thin organic layers (leucine, angiotensin II, and ovalbumin) on silicon, PMMA, and PTFE filters have been provided to the round robin participants. Ions and energies have been chosen for a primary ion velocity of v ~1.0 cm/ns. Secondary ion yields (Y₀) have been measured for the static limit at a fluence of φ_static=10¹⁰ cm^-2 and their evolution recorded for fluences up to φ_static=10¹³ cm^-2. Damage cross-sections, σ, have also been measured and the measurement efficiencies (Y₀/σ) have been determined. An inter-laboratory comparison was performed through these. Insights in best practice for sample contamination and measurement protocol will be provided.

Mass Spectrometry Imaging (MSI) techniques play today an important role for the investigation of molecular metabolic pathways, which are essential for maintaining the cell physiology.

The minimum analyzed area for the chemical imaging using MSI techniques depends mainly on two factors: minimum achievable beam spot size and sensitivity - the total number of molecules that can be detected in this particular beam spot before sample surface is being destroyed. One of the promising MSI techniques is a recently established Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) using MeV ions for the excitation. Contrary to the excitation with keV ions where mostly molecular fragments are detected, MeV ions have the ability to desorb large intact molecules with several orders of magnitude higher yield [1]. Thus, due to the high sensitivity of the MeV TOF-SIMS, molecular composition in the area smaller than 1 µm² can be determined before static limit is reached. Up to now, limiting factor for submicron imaging in our MeV TOF-SIMS system was heavy ion beam spot size of several microns [2]. Here we demonstrate how MeV TOF-SIMS setup can be used for MSI at sub-cellular level using different triggering system, allowing significant reduction in the beam spot size.

In the new setup, trigger for the TOF - START was replaced with a timing signal provided by the Si particle detector used for the Scanning Transmission Ion Microscopy (STIM). Data throughput of the pulse processing electronics (~10 kHz) allowed us to perform measurements with a continuous beam and significantly lower beam current (<1fA). Decrease of the beam current was performed by closing the microbeam object and collimator slit opening, which led to the significant reduction of the heavy ion beam spot size and ion beam divergence using the same focusing elements (Oxford quadrupole triplet). With this new setup the same beam current was used for the MeV SIMS measurements as was in the pulsed ion beam mode, but beam lateral dimension was reduced for more than one order of magnitude.

Capabilities of the new MeV TOF-SIMS setup were demonstrated by measuring chemical composition inside the individual CaCo cells prepared on the thin Si₃N₄ window. The advantage of the present setup is that besides the molecular imaging, STIM image of the cell can be simultaneously recorded providing additional information about distribution of the cell density/thickness.

[1] Y. Nakata,H. Yamada,Y. Honda,S. Ninomiya,T. Seki,T. Aoki,J. Matsuo, Nucl. Instr. Meth. B 267, 2144 (2009)

[2] T. Tadić,I. Bogdanović Radović,Z. Siketić,D. D. Cosic,N. Skukan, M. Jaksic and J. Matsuo, Nucl. Instr. Meth. Phys. Res., B 332, 234 (2014)