Novel multimodal approach of LA-ICP-MS exploring innovative Ru-tetrazene: In situ tracking and bioimaging-guided cancer treatment

Novel multimodal approach of LA-ICP-MS exploring innovative Ru-tetrazene: In situ tracking and bioimaging-guided cancer treatment Kristýna Bilavˇ cíkov´ a a , Michaela Vaˇ sinov´ a Galiov´ a a,* , Roman Hrstka b , Vojtˇ ech Hamala c , Jindˇ rich Karban c a Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno University of Technology, Purkyˇ nova 118, 612 00, Brno, Czech Republic b Research Centre for Applied Molecular Oncology (RECAMO), Masaryk Memorial Cancer Institute, ˇ Zlutý kopec 7, 656 53, Brno, Czech Republic c Institute of Chemical Process Fundamentals, Academy of Sciences of the Czech Republic, Rozvojov´ a 135, Prague 6, 165 02, Czech Republic HIGHLIGHTS GRAPHICAL ABSTRACT •Fast-screening of anticancer Rutetrazene efficacy by single cell LA-ICPMS analysis. •Tracing label-free anticancer agent at femtogram level in cancer cell line. •Multielement imaging of essential metals and Ru-tetrazene by sequential analyser. ARTICLE INFO Handling Editor: Xiu-Ping Yan Keywords: High-resolution 2D imaging Multielemental analysis Quadrupole-based LA-ICP-MS Ru-tetrazene Metalloproteins Single-cell analysis ABSTRACT In this study, we employed LA-ICP-MS to trace a novel, unique Ru-based complex in a lung cancer cell line. This work highlights the importance of the time synchronization of all instrumentation, including the Aerosol Rapid Introduction System. It introduces bulk in situ LA-ICP-MS for rapid screening of chemotherapeutic penetration into the cell and the use of high-resolution 2D single cell imaging, which is used for both large-scale mapping for the monitoring of large cellular variability and for imaging single cells for the precise localization of ruthenium tetrazene accumulation. Furthermore, laser ablation sampling was employed to clarify the mechanism of the kinetics, with Ru-based substance cell influx determined after 6, 12 and 24 h of treatment with the complex. A pilot study employing multielement 2D imaging with Q-based ICP-MS was also conducted, in which the objects of interest were nutritional trace elements whose activity is directly related to the resistance mechanisms of cancer cells. * Corresponding author. E-mail address: [email protected] (M. Vaˇ sinov´ a Galiov´ a). Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca https://doi.org/10.1016/j.aca.2025.344883 Received 16 September 2025; Received in revised form 23 October 2025; Accepted 10 November 2025 Analytica Chimica Acta 1383 (2026) 344883 Available online 11 November 2025 0003-2670/© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). Our results provide essential information about the broad-scale significance of the LA-ICP-MS technique in cancer research, including elucidating treatment efficacy, investigating the synergistic effects of various compounds during treatment and revealing kinetic and resistance mechanisms. These crucial steps towards personalised treatment are demonstrated using novel tetrazene. LA-ICP-MS, which focuses on tracing metals, metalloids and organometallic compounds, has strong potential for implementation in clinical research. 1. Introduction Cancer is a diverse set of insidious diseases with tumour formation and aggressive malignancies causing millions of deaths worldwide each year [1]. The number of cancer patients is growing every year, and although research suggests that a personalised and targeted approach could be the future in oncology, conventional chemotherapy still forms the backbone of cancer treatment. The great chemotherapeutic potential for metal-based agents has been known for platinum derivatives [2]. However, conventional chemotherapeutic Pt derivates [3–5] suffer from poor selectivity, resulting in damage to healthy tissue followed by the development of many negative side effects, and from low efficacy, leading to the use of large doses [6]. Therefore, when designing new anticancer agents, the emphasis is put on selectivity and effectiveness. In the last few decades, many researchers have focused on the synthesis of metal-based compounds replacing platinum with silver, bismuth, iron [7], zinc, gold and copper [8], or osmium, cobalt, rhodium [9], iridium [10] and ruthenium [11–13]. From the perspective of selectivity, the use of metals can be advantageous, as the central metal atom can be reduced to the active form of the compound only at the site of the required effect due to the hypoxic environment of tumours [14]. The metal core is usually also coordinated to individual ligands, which often provide the agent with various subsidiary functions, such as aiding drug penetration into cells and binding to specific targets, etc. Also, complexes whose primary function is antimetastatic are being examined. With regard to investigating the defence mechanisms of cancer cells against conventional chemotherapeutics and potential anti-cancer compounds, as well as to elucidating the initiation, progression, and metastasis of cancer, metalloproteins are currently in the spotlight [15, 16]. The crucial trace elements in metalloproteins, such as iron, selenium, zinc and copper, play several vital roles in the organism [15,17]. Their dysregulation of their levels may be implicated in the development of a number of diseases and/or may be caused by the altered metabolism of damaged cells including changes in the need for trace elements, especially in cancer. Zinc deficiency can reduce the organism’s ability to defend itself against cancer cells [18], as zinc’s anti-tumour activity involves DNA repair or reducing oxidative stress, and zinc deficiency also corresponds to the size and stage of the tumour [17]. Conversely, elevated copper levels lead to cancer development and progression [15], where it is essential for tumour growth as a regulator of signal transduction and gene expression. Moreover, the transmembrane copper homeostasis-related protein copper transporter 1 (CTRL1) is of considerable importance in drug uptake [19]. Research into nutritional elements in the context of cancer brings us closer to a future in which it will be possible to ‘reprogram’ cancer cells and develop tailored-made treatments for each patient. The identification, characterization and quantification of organometallic compounds and elucidation of their interactions with biomolecules are necessary for successful treatment strategies, and it is essential to incorporate cutting-edge advanced analytical techniques. These techniques must be capable of single-cell analysis as well as in situ imaging of the distribution of a potential medicament within cells, as only such information can help to fill current gaps in our understanding of the processes of drug delivery, cellular uptake, and processing in cells, including anticancer activity and targeting at the molecular level. Such techniques will thus play a unique role in improving the agentdevelopment process [20]. Other essential requirements for single-cell analysis techniques are low detection limits, high sensitivity, and the achievement of high spatial resolution for imaging cellular material, as well as the capacity for multielement analysis. In the last decade, a number of powerful analytical tools from the biospeciation toolbox have come to the fore. Synchrotron Radiation-Based X-ray Fluorescence [21, 22], Synchrotron X-ray Fluorescence Microscopy [23,24], Proton Induced X-ray Emission [25,26] and Secondary Ion Mass Spectrometry [27,28] have been used in the past in the context of soft tissues or the 2D imaging of cells. Currently, the gold standard in inorganic investigation is Inductively Coupled Plasma Mass Spectrometry coupled with Laser Ablation sampling (LA-ICP-MS), which is now gaining prominence as a method for the in situ imaging of biological materials. Its undisputed advantages are its simplicity, cost, ability to detect a wide range of elements, high sensitivity, low limits of detection, rapid response, and sufficient spatial resolution. The application of LA-ICP-MS has been verified in the study of neurodegenerative diseases [29] and in the localization of conventional platinum-, clinically tested ruthenium-, and slightly-modified osmium-based drugs in organs and tumours of mice or other animals after treatment [30,31]. In rare cases, the association of essential trace elements linked to cancer progression or response to treatment in human cancer tissues has also been recently investigated [32,33]. The rapid progress of instrumentation has led to a significant increase in spatial resolution and enhanced response allowing the tracking of ultra-trace amounts of analyte at femtogram levels [34–36]. The pilot studies of single cell analysis by LA-ICP-MS are now starting to emerge. However, in most cases, labelling with silver or gold nanoparticles [37,38], or with high concentrations of metal tags or rare earth element-based probes [39] for antibody detection eliminate the risk of undetectability. Moreover, it can help to recognize the target protein specifically through an immune response [40–42]. In addition to the use of labelling, studies have mostly focused on animal material; hence, there is a lack of studies on the monitoring of unlabelled metallodrugs in ultra-trace amounts quantities taken up by human cells; furthermore, the link between metalloproteins and chemotherapeutic agents at the single cell level has not been investigated by LA-ICP-MS. Multielement analysis, i.e. the determination of multiple isotopes within a single in situ LA-ICP-MS measurement, is also challenging, as it is usually limited by the scanning speed of the mass analyser [42]. Sequential mass analysers do not fully support the measurement of multiple elements within a single event, and if multiple isotopes need to be imaged, the dwell time for each analyte isotope must be optimized to achieve the desired detection limits [36]. For this type of analysis, it would obviously be preferable to choose a dynamic time-of-flight (TOF) analyser [43,44]. Nevertheless, TOF analysers are more costly and therefore not as widespread in conventional laboratory workplaces as sequential quadrupole analysers (Q-MS), which are nowadays increasingly used in 2D imaging. In this type of analysis, the sample is continuously scanned, i.e. a dynamic ablative line scan mode is used, which produces a continuous mass flow, however sequential mass spectrometry response. Due to the frequent use of Q-MS and taking into account their limitations, it is necessary to develop a suitable methodology for multielement 2D analysis. The present study deals with the investigation of the possibilities and significance of using the LA-ICP-MS method in oncological research. The main focus is on the scope of information portfolio to be extracted by the application of LA-ICP-MS. It is presented with using a completely unique non-labelled ruthenium tetrazene and the impact of differences in K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 2 harvesting times after treatment of cancer cells with this tetrazene complex. The study thus investigates a) fast-screening in situ bulk analysis by single cell LA-ICP-MS analysis, b) the application of highresolution 2D bioimaging to label-free cell lines for large-format mapping to monitor high variability, and for small-format mapping to monitor the detailed localization of ruthenium complex accumulation, and c) the potential of sequential Q-MS for multielement analysis to explore the involvement of nutritional elements in cancer mechanisms, and to elucidate certain metabolic processes in order to improve the prognosis and treatment of cancer. 2. Experimental 2.1. Cultivation and preparation of cells for solution nebulization ICP-MS analysis All experiments were performed on the human alveolar basal cell adenocarcinoma cell line A549. The cell line was cultured in DMEM medium (Merck KGaA, Darmstadt, Federal Republic of Germany) supplemented with 10 % (v/v) FBS (foetal bovine serum; Gibco, Thermo Fisher Scientific, Inc, Waltham, Massachusetts, USA), 2 mmol/l pyruvate (Gibco, Thermo Fisher Scientific, Inc., Waltham, Massachusetts, USA), 100 U/ml penicillin, and 100 μ g/ml streptomycin (SERANA, Pessin, Federal Republic of Germany). Cells were cultured in a humiditycontrolled incubator at 37 ◦C in a 5 % (v/v) CO 2 atmosphere. Cells passaged a maximum of 20 times were used for the experiment. Subsequent cell preparation was performed separately for laser ablation (tens of thousands of cells) and solution analysis (millions of cells). To prepare pellets for solution analysis, the cell line was cultured in 10-ml culture dishes (TPP Techno Plastic Products AG, Trasadingen, Switzerland) at 60 % confluence. The tested ruthenium tetrazene complex ([ η 5 -(C 5 Me 5 )RuCl-1,4-κ 2 -N,N’-(1,4-bis(2,3,4,6–tetra-O-acetyl-β-Dglucopyranosyl)tetrazene)] (Fig. 1) was prepared at the Institute of Chemical Process Fundamentals of the Czech Academy of Sciences in collaboration with J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences in 2020. The synthesis of this ruthenium tetrazene complex has been reported in our previous study [45]. The ruthenium tetrazene complex contains an attached glucose moiety, which enabled to modulate the lipophilicity of the entire complex by O-acylation. The O-acetylated ruthenium tetrazene showed high single-digit micromolar cytotoxicity to cancer cell lines A2780, SK-OV-3, and noncancerous cell line HEK-293 [45]. We hypothesized that the high cytotoxicity of this complex is associated with its higher lipophilicity and easier penetration into cells by passive diffusion. This motivated us to investigate the biodistribution of ruthenium in cells treated with this ruthenium tetrazene complex. 5 ×10 5 cells were seeded into 10-ml plates. After 24 h, a ruthenium tetrazene compound dissolved in DMSO (Merck KGaA, Darmstadt, Federal Republic of Germany) was added to the cells at a final concentration of 10 μ mol/l. Cells treated with DMSO alone served as a control samples. At each time point (6, 12, and 24 h), the residual culture medium was aspirated from the plates, and the cells were rinsed with 1 ×PBS (0.14 mol/l NaCl (Lach-Ner, s. r.o, Neratovice, Czech Republic), 2.68 mmol/l KCl (Lach-Ner, s. r.o., Neratovice, Czech Republic), 1.47 mmol/l KH 2 PO 4 (Lach-Ner, s. r.o, Neratovice, Czech Republic), and 7.68 mmol/l Na 2 HPO 4 (Lach-Ner, s. r.o., Neratovice, Czech Republic). Cells were subsequently harvested using trypsin (Gold Biotechnology, St. Louis, Missouri, USA). Before centrifugation, cells were counted on a CASY model TT flow cytometer. Then, the cell suspensions were spun down for 10 min at 1000 g and 4 ◦C. The resulting pellets were stored at −80 ◦C until further processing. 2.2. Preparation of cell lines for LA-ICP-MS analysis Glass slides with a diameter of 12 mm (P-LAB, Prague, Czech Republic) were stored in 70 % (v/v) ethanol and illuminated with UV light for 30 min before use. The first day, the slides were placed in 12-well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) into which cells were subsequently seeded at a density of 30,000 cells/ well. Cultivation of the A549 cell line and treatment with ruthenium complex was performed in the same manner as for bulk analysis. The culture medium was aspirated after 6, 12 and 24 h and the cells and slides were rinsed with 1x PBS. Subsequently 0.5 ml of a pre-cooled mixture of methanol and acetone (1:1 (v/v)) stored at −20 ◦C was pipetted into each well of the plate. The fixative mixture was left on the cells for 6 min at room temperature. Thereafter, the slides were removed from the wells and placed onto pulp to air-dry for 1 h. Finally, the slides containing fixed cells were placed in new clean 12-well plates and stored at −80 ◦C. 2.3. Mineralization of cells Mineralization of the samples to determine the average Ru content in the A549 cells was performed using an ultraWAVE microwave device (Milestone Srl, Italy). Pellet samples were moved from Eppendorf microtubes to quartz tubes and weighed on analytical balances with a precision to 0.01 mg. The weights of cell pellets subjected to mineralization ranged from 6.20 to 11.86 mg. The cells were mineralized using 100 μ l of 65 % sub-boiled HNO 3 . The initial pressure of the inert atmosphere of the reactor during cell decomposition was 40 bar. The setting of the instrument did not allow the maximum pressure value to exceed 110 bar. The temperature was ramped over 15 min to 220 ◦C, where it was maintained for 10 min. The total time per decomposition cycle including cooling was approximately 35 min. This procedure yielded a mineralizate, which was diluted by 1 ml of deionised water. 2.4. Solution nebulizer ICP-MS analysis An Agilent 7900 ICP-MS quadrupole ICP mass spectrometer (Agilent Technologies, Inc., USA) in conjunction with an SPS 4 autosampler (Agilent Technologies, Inc., USA) was used to analyse the average Ru content in the cell mineralizates. Optimalization was carried out while monitoring 7 Li + , 87 Y + , 205 Tl + , and 140 Ce + to achieve maximal signal intensities. Plasma robustness was checked via the 140 Ce 2+ / 140 Ce + and 140 Ce 16 O + / 140 Ce + intensity ratios, the ratio intensity values in both cases being less than 5 %. The quantification was established using external calibration with a set of calibration solutions containing an increased amount of ruthenium. The calibration solutions were prepared using a certified reference material – mix of seven elements (Au, Ir, Os, Pd, Pt, Rh and Ru at a concentration of 100 ±0.2 mg/L), part number: AN9087MC-100, Analytical s. r.o., Czech Republic. Calculations were performed using the regression equation y =ax +b. The detection limit was calculated by the instrument from the calibration dependence according to the formula LOD =3⋅SDB a, where SD B represents the standard deviation of the count at the zero-concentration level and a is the slope from the linear equation. The resulting LOD values corresponded to LOD ( 101 Ru + ) =4.05 ng/l. For the solution nebulizer (SN) ICP-MS analysis itself, the final Fig. 1. The structure of the ruthenium tetrazene complex. K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 3 parameters of the analysed 101 Ru + isotope are listed in Table 1. 2.5. LA-ICP-MS analysis To localize the complex accumulation or its adducts in the cell, laser ablation was employed as a sampling method coupled to ICP-MS. LAICP-MS analysis was performed using the Analyte Excite +laser ablation system (Teledyne CETAC Technologies, USA), an ArF* excimer ablation system emitting radiation at 193 nm with a pulse length <4 ns, and the Aerosol Rapid Introduction System (ARIS; Teledyne CETAC Technologies, USA). The LA-ICP-MS was tuned using the standard reference glass material (SRM) NIST 612 to monitor ion intensity and to minimize oxide and doubly-charged ion formation. The fast screening of ruthenium complex levels in cells via bulk in situ analysis was performed using LA-ICP-MS, where 40 cells were analysed separately for each sample. The diameter of the laser beam was increased to 25 μ m to ensure that the entire cell surface in the x and y axes was always ablated (average cell size approximately 18 μ m). At the same time, the dose was set to 5 laser pulses per point to ensure complete ablation in the z-axis. For 2D imaging of a larger sample area, and thus for the analysis of a larger number of cells, the dynamic ablation line scan mode was chosen. An important consideration in the monitoring of ruthenium tetrazene distribution within a single cell is to achieve as high a spatial resolution as possible. Therefore, the beam diameter was set to 2 μ m. The scan speed was adjusted in dependence on a frequency of 50 Hz [40], and, finally, the higher value of the scan rate was chosen – specifically, 50 μ m/s. To sputter whole cellular material, and concurrently to avoid ablation of the glass substrate, the sampling was achieved with 2.5 J/cm 2 fluence. To study the precise localization of the ruthenium complex within the cellular system using 2D imaging, a smaller area containing 3–5 cells was also ablated, with the emphasis on visualizing ruthenium within a single cell, using the ablative mode of spot analysis employing a line of spots pattern. To investigate the correlation between the distribution of the ruthenium tetrazene and the essential elements copper and zinc in cancer cells. Due to the need for slower material sputtering in multielemental sequence detection Q-MS, a lower scan speed of 17 μ m/s was chosen. The SRM NIST 610 was analysed simultaneously with the sample to correct for instrumental drift and instrument sensitivity changes. The set-up conditions for the laser ablation system had to be adjusted for NIST 610; due to the variability in the homogeneity of this material, the laser beam diameter was increased [46]. Due to the absence of ruthenium in NIST 610, the signal response of the 85 Rb + isotope and 28 Si + was monitored, since NIST is a glass material. The resulting parameters for all LA-ICP-MS experiments, in situ bulk analysis, and 2D imaging, including multielement determination, are shown in Table 2. The HDF-based image processing software HDIP (Teledyne Photon Machines Inc., Bozeman, MT, USA) was used to evaluate all data acquired by LA-ICP-MS. In the case of spot analysis, the resulting signal peak areas corresponding to the ablated cells were integrated using HDIP software after background correction, and these values were corrected to the cell area, which was calculated approximately from the diameter of each cell by assuming a circular shape. To evaluate the 2D data, the same software was used to visualize the results when the isotope images were reconstructed. 3. Results and discussion 3.1. In situ bulk analysis via LA-ICP-MS Due to the need for a dose-response characteristic, the efficient screening of ruthenium compound kinetics and its levels in cells was performed using in situ bulk LA-ICP-MS analysis. The advantage of this Table 1 SN-ICP-MS working parameters. ICP-MS parameters Analysed isotopes 101 Ru + Integration time (ms) 100 RF power (W) 1550 Sample depth (mm) 8.0 Nebulizer gas (l/min) 1.03 140 Ce 2+ / 140 Ce + 0.79 % 140 Ce 16 O + / 140 Ce + 0.96 % Table 2 LA-ICP-MS working parameters for bulk analysis, 2D imaging of 101 Ru + and 2D multielemental imaging. ICP-MS parameter In situ bulk analysis 2D imaging 2D multielemental imaging NIST 610 Analysed isotopes 101 Ru +63 Cu + , 66 Zn + , 101 Ru +28 Si + , 85 Rb + Integration time (ms) 30 90, 90, 90 30, 10 RF power (W) 1550 Sample depth (mm) 5.5 Nebulizer gas (l/min) ~0.85 LA parameter In situ bulk analysis 2D imaging 2D multielemental imaging NIST 610 Ablation mode spot analysis line scan line of spots line scan line scan Spot size ( μ m) 25 2 2 2 8 Fluence (J/cm 2 ) 2.5 2.5 2.5 2.5 2.5 Scan speed ( μ m/ s) –50 –16.5 50 Shot count (−) 5 –5– – Rap rate (Hz) 50 50 50 50 50 Line distance ( μ m) –2 2 4 – Spot distance ( μ m) – – 2– – Line length ( μ m) –750 85 400 3000 Number of lines (−) –375 43 100 1 He low rate MC1+MC2 (l/ min) 0.6 Fig. 2. Box plot analysis of the average composition of samples with different times of cell treatment. K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 4 method consists in the rapid screening of complex efficacy and the elimination of a mineralization step as in SN-ICP-MS. The experiment using the spot analysis mode of laser ablation was realized with 25 μ m laser beam must be set to ablate the whole cell as the analysed material is in diameter of approximately 18 μ m. As the bulk analysis determines the average response of ruthenium corresponding to the individual cell without rastering, each peak in the time-resolved signal relates to an individual cell. The analysed cell samples were placed on glass slides and collected at 6, 12 and 24 h of treatment of the cells with the complex. Individual cells were analysed under the working conditions for spot analysis (Table 2), with a laser beam size of 25 μ m and 5 pulses to ablate the entire cell volume within a single spot. For each sample, 40 cells were sputtered. A control sample without treatment was also examined and no 101 Ru + isotope signal was detected. The resulting integrated peak areas corrected for cell area are summarized in Fig. 2. The results from cells collected after 6 h provide a dataset with asymmetry, where most of the data is concentrated in the lower values; however, there are some extremely high values that increase the average. The difference between the median value equal to 83 (illustrated by the horizontal line inside the box) and the mean value 93 confirms the asymmetric distribution of the data. Moreover, the large interquartile range suggests considerable variability. The data corresponding to the 12-h treatment are more consistent, providing a data set with a symmetric distribution and a distribution around a midpoint value of 129. The large interquartile range indicates considerable variability in the data, but consistency across the entire data set. The results of the 24-h treatment show data with significant right asymmetry and large variability. Most of the data are concentrated in the lower values around a median of 78, which is significantly lower compared to the mean value of 104. According to the response-dose characteristic, it can be assumed that after ruthenium tetrazene administration, cellular influx occurs, represented by a sample harvested after 6 h with considerable system variability. The cell sample after the 12-h treatment most likely represents the equilibrium of the system; i.e., the saturation of the cells and the beginning of the efflux, which is in a good agreement with the symmetric distribution of the data. After 24 h, the values are at the same level as those for the sample after 6 h. Thus, it can be expected that cellular efflux predominates, as indicated by the large variability of the system. Variation within individual samples and in cellular uptake and efflux might be due to differences in cell design as well as cell transformation after 24-h exposure and an increase in the number of nonviable cells with the original amount of ruthenium complex. These cells are no longer subject to influx or efflux. The average composition is often determined using solution ICP-MS analysis after mineralization of the cell pellets. Therefore, this approach was also used to compare the LA-ICP-MS results (Table 3). The results of the solution analysis of the cell pellets after mineralization showed that the ruthenium level in the control sample was below the detection limit, which was calculated from the calibration dependence of 2.6 fg per cell. This is in agreement with the results of the LAICP-MS analysis. The highest ruthenium accumulation was identified after the 6-h treatment. Approximately two times lower levels were found in cell samples collected 12 and 24 h after treatment. Although the ruthenium contents in these two samples do not overlap when standard deviations are taken into account, the standard deviations in the case of SN-ICP-MS represent only variations in measurement. SN-ICP-MS did not fully confirm the trend of in situ bulk LA-ICP-MS analysis. The discrepancy could be caused by the different cell numbers used in the preparations for SN (millions of cells per pellet) versus LA (30,000 cells per glass, 40 cells analysed) analysis. The laser ablation results show a higher amount of Ru in cells with a 12-h treatment, and a similar response with 6-h and 24-h treatments. Furthermore, the bulk in situ analysis exhibits larger scattered values than the standard deviations of the solution analysis presented in Table 3. The standard deviations of the solution analysis are calculated from repeated measurements of a single replicate sample, whereas the scatter of the laser ablation results corresponds to the variability of individual cells. Another discrepancy between LA and solution analysis is the process of sample processing. In the case of solution analysis, the cells were spun down into an Eppendorf vessel containing a known number of cells, which were counted using a cytometer. Subsequently, the cell suspension with the known concentration of cells must be transferred into the reaction vessels of the digestion system by pipetting, in order to replace the maximum original number of cells. The final number of cells to be digested is calculated proportionally according to the sample weight. During this process, errors can be caused by an unknown exact weight of the residual medium in the cell suspension, as the total weight is attributed to the cells only. However, we suppose that the main reason for the slightly different results of LA and SN relates to working with a completely different number of cells: digestion and solution analysis of millions of cells versus 40 cells analysed by LA-ICP-MS. The different cell numbers could result in different levels of ruthenium complex availability to the cells themselves. Moreover, the laser ablation is burdened with manifestation of the high variability of cells more than solution analysis ICP-MS. The results could be comparable only if more than 40 cells are investigated. Although it is not possible to define the maximum content for a specific treatment time using significance statistics due to high scattering, in situ bulk analysis can be used to differentiate between cells based on their cargo, or to determine the efficacy of treatments involving different types of compounds, where greater differences in influx are expected. 3.2. 2D imaging by line scanning mode In addition to in situ bulk analysis, large-scale 2D cell mapping for rapid localization was performed. Localization is essential for evaluating treatment interactions, such as whether it reaches the nuclei or accumulates in the intracellular space. It requires to achieve as high spatial resolution as possible, therefore, the beam diameter was set to 2 μ m. The samples were sputtered in line scanning mode; however the drawback of that dynamic sampling mode is the overlapping of ablation craters depending on the scan speed and frequency. On the other hand, a suitable choice of parameters leads to a smaller depth of the ablated trace. The scan speed 50 μ m/s settings in combination with a frequency of 50 Hz resulted in a resolution of 1 laser pulse for each 1 μ m. The goal was to ablate, in a single analysis, as many cells as possible with respect to monitoring variability in ruthenium tetrazene levels as a function of cellular influx. For the purpose of high-resolution 2D imaging, it was necessary to synchronize the laser ablation system with the mass spectrometer, as the introduced Aerosol Rapid Introduction System (ARIS) enables a lower washout time than the conventional HelEx II ablation cell. These devices together allow the entire ablation cell volume to be washed out in the order of units to tens of milliseconds (<20 ms [47]), whereas without ARIS, the washout would take higher hundreds of milliseconds (≈700 ms, [48]). Rapid cell washout has many advantages, primarily related to improved spatial resolution, and thus results in a narrow peak corresponding to the aerosol from a single laser pulse with no gradual peak smearing. The fast washout also minimizes aerosol mixing between pulses, allowing detection of individual ablation events Table 3 Average amount of Ru per cell exposed to ruthenium tetrazene, harvested at different time intervals. Sample Ru content (fg/cell) Control <2.6 6 h after treatment 454 ±15 a 12 h after treatment 234.6 ±9.6 a 24 h after treatment 254 ±14 a a Standard deviations based on replicate sample measurements (n =3). K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 5 by the mass spectrometer. The signals are also more “concentrated” in time, i.e., they are characterized by higher peak intensities and lead to an improved signal-to-noise ratio in ICP-MS [49]. Proper synchronization of the laser ablation system with the mass spectrometer is crucial for obtaining correct information on the composition of the aerosol. In this context, a detailed study of the effect of the integration time (IT) of the monitored isotope on the time-resolved ICP-MS response was performed. The study of the integration time effect was first performed on the A549 cell model, where the 101 Ru + isotope was monitored under the line scan conditions shown in Table 2. Due to the huge variability of the living system, no reproducible trend with decreasing integration time during cell ablation was demonstrated. The increase and/or decrease in intensity values were more related to the analyte content in the individual cells as well as to their design, such as their size and topography, rather than to the change in integration time itself. Therefore, the investigation was subsequently performed on NIST 610 standard reference material and the response to 85 Rb + was monitored due to the absence of ruthenium in the reference material. Rubidium is in NIST 610 at the relatively high content of 425.7 μ g/g [50]. The setup conditions for NIST 610 laser ablation were modified due to the different physicochemical properties compared to cellular material, including a higher threshold energy required to achieve ablation; thus, the fluence was increased to 3 J/cm 2 [51]. In addition, due to the variability in the homogeneity of this material [46], the laser beam diameter was increased to 8 μ m. The final and important parameters influencing the laser ablation process were retained – specifically, a 50 Hz frequency and a 50 μ m/s scan speed. The effect was monitored in the ablation mode of the line scan for integration times ranging from 100 to 10 ms in steps of 10 ms, and 3 ms. The time-resolved signals obtained by the ablation of a single line of 80 μ m in length give different waveforms depending on the value of the integration time, Fig. 3. The 85 Rb + isotope signal is stable from 100 ms, when the analysed aerosol is highly mixed. Even at the lower integration time of 20 ms, a relatively stable signal is achieved, but it displays more fluctuations and the number of points rendering the time-resolved signal increases. A completely different signal behaviour is visible at an IT of 10 ms, when the signal oscillates around a mean value with gradually increasing and then decreasing intensity, resulting in a specific signal clustering structure. This is probably caused by the resynchronisation of the LA washout and ICP-MS detection. In contrast, an IT equal to 3 ms shows that the signal drops to background level every 20 ms. The total number of maxima is equal to 80, which corresponds to the number of pulses required to ablate an 80μ m length line at a scan speed of 50 μ m/s and a frequency of 50 Hz. Thus, it can be concluded that under the given experimental conditions and using ARIS, it is possible to determine the aerosol composition from each laser pulse. A noticeable difference is also the change in the intensities achieved. Within the course of the time-resolved signal from 100 ms to 20 ms, the achieved intensity values range from 9.0 ×10 4 –10 5 cps. Up to 10 ms, due to signal oscillation, the maximum achieved intensities are observed to reach above 1.5 ×10 5 cps and within the lowest integration time, to reach almost 3.0 ×10 5 cps. This would suggest that despite the rapid washout and analysis of material from a single laser pulse, the resolution of ICP-MS is enhanced. In a study of the synchronization of a laser ablation system with ARIS and a quadrupole mass spectrometer, it was shown that by reducing the integration time to units of ms, an excellent and fast washout of the aerosol obtained by the ablation of homogeneous NIST 610 material containing high levels of 85 Rb + analyte is achieved. However, the use of this integration time for 2D mapping is limited by the length of the analysis in terms of the computational capabilities of the Q-based ICP mass spectrometer. The ICP mass spectrometer is typically capable of processing only a limited volume of data at an IT of 3 ms, corresponding to approximately 25 min of measurement time. For these reasons, the IT value for the 101 Ru + isotope was set to 30 ms. It was experimentally verified that at this IT no beats formation in the time resolved signal occurs and that detailed cell rendering is achieved. In this way, it is possible to localize elements in small cellular compartments. The aim of this part of the experiment was to rapidly localize the ruthenium complex using large-format 2D imaging while investigating cellular influx, efflux and cell saturation, i.e. to determine the overall kinetics of ruthenium tetrazene in the cellular compartment by means of LA-ICP-MS. The individual cell samples differed in the time of RuFig. 3. Time-resolved 85 Rb + signal in NIST 610 at various integration times: a) 100 ms, b) 30 ms, c) 10 ms, and d) 3 ms. K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 6 compound exposure – specifically, 6, 12 and 24 h. The fitted parameters (Table 2) allowed an area of 750 ×750 μ m containing approximately 150 cells to be investigated, and thus the variability of the living system to be studied in a single analysis, Fig. 4. A control sample that was not treated with the ruthenium complex was also analysed along with the samples. It was verified that no 101 Ru + signal was detected in the control sample (not shown). In Fig. 4, for all cell collections at time intervals of 6, 12 and 24 h of ruthenium tetrazene treatment, the localization of the 101 Ru + isotope in the cells as well as in the vicinity of cells is clearly defined. In all the cells located in the analysed area after 6-h treatment in Fig. 4 a), ruthenium, and therefore the complex or its adducts or metabolites, were present in different levels. Due to the absence of ruthenium in the SRM, it was not possible to quantify these abundances Fig. 4. Images of the analysed area from the CCD camera of the laser ablation system (left) and the 101 Ru + isotope distribution map (right) obtained by linear ablation of the A549 cell line after treatment with ruthenium tetrazene with a harvesting interval of a) 6 h, b) 12 h, c) 24 h. The scale bars placed in imaging maps correspond to the CCD images. K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 7 Fig. 5. Images of the analysed area taken by the CCD camera of the laser ablation system (left) and the distribution maps of the 101 Ru + isotope (right) in the A549 cell line after 6-h treatment obtained by spot analysis. The scale bars placed in imaging maps correspond to the CCD images. K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 8 Fig. 6. Images of the analysed area taken by the CCD camera of the laser ablation system (left) and the distribution maps of the 101 Ru + isotope (right) in the A549 cell line after 12-h treatment obtained by spot analysis. The scale bars placed in imaging maps correspond to the CCD images. K. Bilavˇ cíkov´ a et al. Analytica Chimica Acta 1383 (2026) 344883 9