AFM-optimized single-cell level LA-ICP-MS imaging for quantitative mapping of intracellular zinc concentration in immobilized human parietal cells using gelatin droplet-based calibration

The goal of this study was to develop and validate a robust, reproducible calibration strategy for quantitative LA-ICP-MS imaging of intracellular zinc at the single-cell level. To address the challenge of calibration in elemental bioimaging, the researchers introduced a gelatin-based matrix with a homogeneous Zn distribution, eliminating the need for biological reference standards and reducing analysis time.

Laser ablation conditions were optimized using atomic force microscopy (AFM) to ensure accurate volume measurements and reproducibility. The method was successfully applied to Zn-treated human parietal cells, providing high-resolution spatial maps of Zn distribution. Its accuracy was confirmed by comparison with bulk ICP-MS measurements, demonstrating the method’s potential for broader biological and biomedical applications.

The original article

AFM-optimized single-cell level LA-ICP-MS imaging for quantitative mapping of intracellular zinc concentration in immobilized human parietal cells using gelatin droplet-based calibration

Valerie Boger, Philip Pirkwieser, Noreen Orth, Melanie Koehler, Veronika Somoza 

Analytica Chimica Acta, Volume 1355, 2025, 343999

https://doi.org/10.1016/j.aca.2025.343999

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

For the replacement of biological material, various calibration strategies have been proposed. These include, for example, polymer-based approaches, like polymethylmethylacrylate (PMMA) films on quartz substrates [12] or cold-curing resin blocks spiked with different amounts of the metal of interest [13]. Sela et al. [14] used a sol-gel matrix produced by tetraethyl orthosilicate (TOES), and Turková et al. [15] tested agarose gel as a matrix similar standard. Another study evaluated natural materials, such as egg yolk, to quantitate the distribution of thulium in different tissues, achieving good linearity and detection limits [16]. Ink-printed standards, using commercially available printers with gold thin-layers as an internal standard for matrix-related ablation differences, have been developed as well [17]. Resembling the composition of biological material, gelatin has proven to be a good alternative as an external calibrant [11]. Due to the high protein content, it resembles the protein-rich cellular material and has shown similar particle transport properties as for biological material [18]. Gelatin sections [2,19,20] as well as gelatin droplets [21,22], micro-array standards [23] and micro-droplets based on micro-spotting [11,24], have been widely tested as calibration standards for LA-ICP-MS. In droplet-based approaches, the element distribution can be inhomogeneous due to the “coffee-stain” and/or the “Marangoni” effect, caused by chromatographic effects [25] and a concentration and/or temperature gradient upon drying [26]. Šala et al. [27] showed that these effects can be circumvented by certain drying conditions of the gelatin droplets. However, each element behaves differently in the gelatin matrix, so the preparation described in literature often only works for a limited number of elements. Westerhausen et al. [2] compared matrix-matched calibration with tissue homogenate standards, spiked with the elements of interest, with an approach based on gelatin standards. The ablation of homogenized brain standards resulted in significant signal drifts as a consequence of uncontrolled ablation due to heterogeneity in surface and structure. In contrast, using gelatin yielded improved ablation characteristics, homogeneous element distributions and reproducible physical properties [2]. Another possible way of overcoming the problem of heterogeneous elemental distribution is the ablation of the entire droplet [21,22]. Van Acker et al. [22] compared the ablation of the entire droplet with spot ablation of homogeneous air-dried gelatin droplets. The results of both approaches were in good agreement. As the ablation of an entire droplet becomes time consuming with larger droplet volumes, arrays of gelatin micro droplets of 300–500 pL in volume were produced with the help of micro-spotting devices [11,24]. Overall, it is difficult to find one unified fitting approach, since each sample requires compound-specific ablation settings.

In this study, a method of gelatin standard matrix-matched sample preparation and calibration procedure that can be applied for different biological samples was evaluated. The basic idea was to avoid handling biological material by using a matrix replacement with a composition similar to the sample, so that errors due to different ablation behavior can be reduced to a minimum. Within the gelatin matrix, the element to be investigated should also be homogeneously distributed. Thus, the ablation of the standards in their entirety would no longer be required, thereby reducing the analysis time for the calibration via LA-ICP-MS. The HGT-1 human gastric cell cancer line was selected upon zinc exposure as zinc represents an essential trace element playing a key role for several important biological processes in the human body, also being absorbed via the intestinal epithelium.

2. Materials and methods

2.3. LA-ICP-MS instrumentation

All laser ablation experiments were performed with an Iridia 193 nm laser ablation system (Teledyne CETAC Technologies, NE, USA), coupled to a NexION 5000 multi-quadrupole ICP-MS instrument (PerkinElmer, MA, USA). The laser ablation system was equipped with a Z-prime cobalt long pulse ablation cell and the Aerosol Rapid Introduction System (ARIS). Helium was used as a carrier gas (0.3 L/min) for aerosol transport from the ablation cell to the ICP system. The measurement conditions were fine-tuned daily, using the NIST SRM 612 glass certified reference material (National Institute of Standards and Technology, MD, USA). The nebulizer gas flow was optimized from 0.82 to 1.00 L/min, generating maximum ¹⁴⁰Ce ⁺ signals, low oxide formation based on ²³²Th⁺¹⁶O⁺/²³²Th⁺ (<1 %) and low elemental fractionation based on the ²³⁸U⁺/²³²Th ⁺ ratio (∼1). The Quadrupole Ion Deflector (QID) parameters were fine-tuned by adjusting the lens scanning system for maximum efficiency, measuring ⁷Li⁺, ²⁴Mg⁺, ¹¹⁵In⁺, ¹⁴⁰Ce⁺, ²⁰⁸Pb⁺ and ²³⁸U⁺ as representatives of the entire mass range. In all experiments ∼20 s before the laser was fired, data was recorded to create a baseline level with the gas blank signal. The integration and read-out rate were synchronized to match the laser ablation repetition rate. The ICP-MS signals, received from Syngistix v.3.3 (PerkinElmer, MA, USA) were synchronized with the timestamps in the laser log files from Chromium v.3.1 (Teledyne CETAC Technologies, NE, USA) and further visualized via HDIP-v1.8 (Teledyne CETAC Technologies, NE, USA).

2.5. Atomic force microscopy

Atomic force microscopy was performed with a BioAFM NanoWizard V (JPK BioAFM – Bruker Nano GmbH, Berlin, Germany) to determine the exact ablated volume in a gelatin droplet as well as in a single HGT-1 cell. For this purpose, prior the experiment, an area of 25 μm² (corresponding to the used spot size of 5 μm square shape for the LA-ICP-MS imaging experiments) was quantitatively ablated from a single cell with laser ablation as explained earlier. Additionally, gelatin was ablated, using different dosages ranging from 1 to 10 shots per position with a laser fluence of 0.53 J/cm². Integration and read-out rate were synchronized to match the laser ablation repetition rate. Subsequently, areas of varying size and resolution were scanned by operating the AFM in tapping mode in air using TESP-V2 cantilevers (Bruker, CA, USA; calibrated spring constant of 40.1 N m⁻¹) with a driving amplitude of 0.14–0.21 V. This way, the average cell height of various dried HGT-1 cells on a microscope slide was also determined. The topography images were evaluated with the AFM data analysis software Gwyddion v2.62.

3. Results and discussion

3.1. Gelatin matrix optimization

Elemental distributions in the gelatin matrix differentiated substantially under the same preparation conditions, depending on the examined metal. Fe integrated well into the gelatin matrix through stabilizing and/or complex building properties, whereas Zn and Al were not distributed homogeneously within the same droplet (Fig. S1). Re fundamentally yielded a more homogeneous distribution compared to Zn. In addition, a higher temperature during drying had a positive impact on the homogeneous distribution of both Re and Zn (Fig. S2). From these results, we concluded that metals behave differently in the gelatin matrix, due to chromatographic effects, where weight and size of the corresponding element play a role, as well as stabilization within the matrix by other elements, and complex-building effects. For those elements cross-linking with gelatin structures, which leads to immobilization of the respective element. These cross-linking elements include cations like Al³⁺, Cr³⁺, Fe³⁺, Ce³⁺, La³⁺, Pt²⁺, etc. [30] Since only these elements have been used in some studies so far, the inhomogeneity was not noticeable [31].

3.2. Optimization of the laser parameters in combination with AFM

Van Acker et al. [32] showed diffraction effects influenced ablation thresholds for spot sizes <3 μm, resulting in increased values. Ablation thresholds of ∼20–40 mJ/cm² for human tissue have been reported for an 193 nm ArF excimer laser [33]. Van Malderen et al. [23] reached a quantitative removal of gelatin at a 0.7 J/cm² energy density, while a small amount of glass was removed, as observed via the ²⁹Si ⁺ signal. Schweikert et al. [11] chose a fluence of 0.60–0.90 J/cm². The topography image measured via AFM (Fig. S6) shows unwanted thermal effects, using a laser energy of 1.00 J/cm². Hence, a lower laser energy density of 0.53 J/cm² was chosen for the LA-ICP-MS calibration in this study. Fig. S6 demonstrates the linear relationship between the ⁶⁶Zn ⁺ signal intensity with increasing dosage. Since the dried gelatin standards were found to be thicker than a cell, the right dosage needed to be found for the calibration, quantitatively ablating the cells, but not too much of the gelatin. Moreover, the laser focus must also be considered, which can be difficult to adjust due to the slight curvature of the gelatin droplet surface. Therefore, an even area of the droplet should be selected to minimize the influence of different heights of the droplet. Overall, taking into consideration the natural differences in cell size and thickness, it is difficult to find the perfect dosage and it will always rely on finding the most suitable compromise. To determine the exact ablated volume in a gelatin droplet as well as in a single HGT-1 cell, AFM was used as an addition to LA-ICP-MS. The volumes of the craters ablated via LA-ICP-MS were analyzed using AFM and calculated as follows: Estimated volume: 5 μm × 5 μm x height [μm]. This was done for a series of 10 craters, using different dosages ranging from 1 to 10 shots per position. Additionally, the ablated volume of an area of 25 μm² in a single HGT-1 cell was calculated, using a cell height of 1.17 ± 0.10 μm (average cell height for 10 dried HGT-1 cells, determined via AFM). The height-profiles and corresponding AFM-topography image for the via laser ablated craters in a single HGT-1 cell, as well as in gelatin with a dosage of 9 shots per position are shown in Fig. 3. For the LA-ICP-MS calibration, a dosage of 9 shots per position was chosen, since the ablated volume in the gelatin achieved nearly the same as in an average cell (29.7 ± 2.45 μm³, Fig. 4). The results showed a good linear fit (R² = 0.9931) between the crater volumes and the corresponding laser dosage, shown in Fig. 4.

Analytica Chimica Acta, Volume 1355, 2025, 343999: Fig. 3. Depth profiles in a single HGT-1 cell (A) and in gelatin (B) and the corresponding ablation craters (right). LA-Parameters: fluence: 0.53 J/cm2; spot size: 5 µm square shape; dosage: 9 shots per position.

4. Conclusion

This work demonstrated an LA-ICP-MS imaging approach, optimized by AFM, to quantitatively investigate cellular Zn uptake at a high spatial resolution. The gelatin-based calibration proved to be a suitable replacement for a calibration based on biological samples, allowing easy and flexible sample preparation, with high potential for its application to a wide range of other metals.