Research Projects

Abstract

The main objective of the project is to develop a new MeV-SIMS molecular imaging method that would enable the imaging of heavy molecules (above 1 kDa) at the submicron and subcellular levels. Such a powerful molecular imaging method would be of particular interest in fields such as medicine, biomedicine, and biology, where the spatial distribution of biomolecules still cannot be adequately measured, despite the highly valuable information it provides.

Secondary ion mass spectrometry (SIMS) is one of the most widely used molecular imaging methods. It is known as the technique offering the highest spatial resolution, reaching values as low as 50 nm when monoatomic metal ions are used. On the other hand, SIMS is also known for the destructive nature of the secondary-ion sputtering process, which results in a high degree of molecular fragmentation. At the same time, the fraction of ionized molecules is very low (approximately 10⁻⁵). These problems have been partially addressed by using larger clusters as primary ions, since the energy of the primary ion is distributed among the atoms within the cluster. Although this approach has partially mitigated the limitations of SIMS, the generation and use of such ions have had a substantial negative impact on the spatial resolution of the method.

An alternative approach is the niche MeV-SIMS technique, in which the sample is bombarded with ions having energies in the MeV range rather than the keV range used in conventional SIMS. The use of faster ions also changes the desorption process: as the energy of the primary ions increases, the dominant energy-loss mechanism gradually shifts from nuclear stopping toward electronic excitation. This so-called electronic stopping process is much gentler on the sample and causes considerably less fragmentation. The use of fast ions has also been shown to enable the desorption of very heavy molecules with masses exceeding 10 kDa. However, manipulating such high-energy ions is technically very challenging, which makes focusing the ion beam particularly difficult.

Within the project, we intend to develop a routinely accessible mass spectrometry imaging method in which MeV-energy ions would be generated using a sputter ion source of an ion accelerator and subsequently accelerated to their final energy. The beam would then be reduced to a very low intensity and delivered continuously to the target. This approach would enable the beam to be focused to a spot size of approximately 500 nm to 1 µm. The primary ions would then be detected via the secondary electrons generated upon impact. Once an electron is detected, the voltage applied to the sample would be raised, causing the positive secondary ions to be accelerated into a time-of-flight mass spectrometer. The electron signal would also serve as the “start” signal for the time-of-flight measurement, while the signal from the MCP detector at the end of the mass spectrometer would provide the “stop” signal.

We also intend to develop a modern sample-preparation method that would enable submicron imaging of intact cells, and the capabilities of the method will be demonstrated through the analysis of plant tissues. To further increase the secondary-ion yield, we will also investigate different sample-preparation approaches that have proven effective in conventional SIMS analyses for improving the overall efficiency and sensitivity of the method.

Researchers 

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IJS:

• dr. Boštjan Jenčič
• dr. Mitja Kelemen
• prof.dr. Primož Pelicon
• doc dr. Paula Pongrac (from 1.1.2026)
• Mirjana Sepahyar Lorentzen
• dr. Primož Vavpetič

BF:

• Valentna Bočaj
• Žan Cimerman
• Doc. Dr. Matevž Likar
• Ana Mezinec (from 1.1.2026)
• Dr. Jure Mravlje
• Doc. dr Paula Pongrac
• Prof. dr. Marjana Regvar
• Tanja Sunčič (until 31.12.2025)
• Dr. Katarina Vogel Mikuš

The phases of the project and their realization

Phase 1: Efficient Detection of Continuous Primary Ions

The arrival of a primary ion at the sample will be detected via secondary electrons (SE). A CEM detector will be positioned in front of the sample, next to the entrance of the mass spectrometer. In WP1, we will optimize the distance and angle between the detector and the target, as well as the voltage applied to the detector. Our goal is to achieve close to 100% detection efficiency for primary ions while eliminating any undesirable perturbations caused by the CEM detector.

The primary ion current will be measured using a second CEM detector positioned along the primary ion beam axis, and the detection rates of the two detectors will then be compared. In addition, mass spectra will be measured with and without the additional CEM detector for secondary-electron detection, allowing us to evaluate and optimize its configuration.

Phase 2: Efficient Transport of Secondary Ions

Following desorption, secondary ions have kinetic energies ranging from 0 to 10 eV, most commonly around 5 eV. This means that they can be accelerated into the mass spectrometer after approximately 150 ns, although the exact time also depends on their mass, with heavier ions remaining near the sample for longer periods.

Since we intend to apply a voltage of approximately 5 kV only after detecting secondary electrons, very little time is available for signal processing. The negative signal from the CEM detector must first be amplified and subsequently converted into a positive pulse with a duration of approximately 3–5 µs, corresponding to the time required to extract all secondary ions into the mass spectrometer. A dedicated signal converter will therefore be designed and constructed to enable sufficiently rapid processing and conversion of the signal into the required form.

Phase 3: Development of Frozen-Tissue Preparation and a MeV-SIMS Measurement Protocol for Routine Analysis

We will collaborate with the Biotechnical Faculty of the University of Ljubljana. The objective is to develop a routine method for preparing frozen tissue samples. Samples will be sectioned to thicknesses ranging from 10 to 60 µm using a cryotome and subsequently frozen in liquid nitrogen to minimize the formation of ice crystals within the cells.

The frozen samples will then be introduced into the experimental chamber, where they will be cooled through thermal contact with a liquid-nitrogen reservoir. We also intend to design and construct a dedicated sample holder that will allow a high voltage to be applied to the target while simultaneously providing high thermal conductivity.

Biological tissues will subsequently be analyzed to investigate the effects of abiotic stressors, such as salts and heavy metals, on chemical processes within the tissues by mapping changes in the spatial distribution of biomolecules.

Phase 4: Further Enhancement of Secondary-Ion Yield

Secondary-ion yields are typically reported for reference samples containing relatively high concentrations of the molecules of interest. Biological tissues, however, contain a very large number of different biomolecules, many of which are present at relatively low concentrations. To gain a more comprehensive understanding of biological processes, we also aim to map these low-abundance biomolecules. This will most likely require sample-preparation protocols that substantially increase the secondary-ion yield.

Three principal approaches will be investigated: carbon deposition, deposition of metals such as silver and gold, and the addition of small quantities of salts. Initially, their effects will be evaluated using reference standards of arginine, cholesterol, and polyethylene glycol (PEG) with molecular weights of 600, 1,000, 2,000, and 3,000 Da.

We will subsequently investigate the effects of these treatments on biological tissues, assessing how such sample modifications influence molecular imaging quality and determining the extent to which the treatments alter the samples themselves.

References

Lorentzen et al., 2025: The Static Limit in MeV Secondary Ion Mass Spectrometry, External link to https://doi.org/10.1021/jasms.5c00024 Open in new window

Regvar et al., 2025: Correlative Imaging of Structural Biochemistry in Plant and Food Quality Research Within and Interoperable Data Acquisition Platform, External link to https://doi.org/10.1093/mam/ozaf057 Open in new window