Advancing elemental analysis by collision/reaction cell technology and micro-droplet calibration for bioimaging applications by LA-ICP-TOFMS

In the study published in the Analytica Chimica Acta journal, researchers from the University of Vienna, Austria evaluated the use of collision/reaction cell (CCT) mode in laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) for comprehensive elemental analysis in bioimaging applications.

The CCT mode demonstrated superior performance over standard mode, particularly for endogenous elements prone to interferences, enhancing sensitivity for high-mass elements by 1.5–2 times without compromising endogenous element detection. Accurate quantification of elements like iron and selenium in serum reference materials confirmed the method's reliability. By streamlining analytical workflows and eliminating extensive post-data processing, this approach advances bioimaging applications, ensuring robust, validated measurements across the entire mass range.

The original article

Advancing elemental analysis by collision/reaction cell technology and micro-droplet calibration for bioimaging applications by LA-ICP-TOFMS

Sarah Theiner, Elisabeth Foels, Gunda Koellensperger

Analytica Chimica Acta, Volume 1332, 2024, 343345

https://doi.org/10.1016/j.aca.2024.343345

licensed under CC-BY 4.0

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

Highlights

• Evaluation of collision/reaction cell technology for bioimaging applications by LA-ICP-TOFMS.
• Addressing analysis of elements across the entire mass range by LA-ICP-TOFMS.
• Incorporation of micro-droplet containing serum reference material in bioimaging workflows.
• Micro-droplet calibration and quality control in one workflow.
• Improved limits of quantification, isotope ratio precisions and accuracies for the CCT mode.

Abstract

Background

Bioimaging applications by laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) require comprehensive elemental analysis spanning the entire mass range. Within biological systems, endogenous elements are critical for maintaining metal homeostasis in cells, a fundamental aspect in disease progression when disrupted. Additionally, elements from the higher mass range may originate from various sources such as metal-tags for immuno-mass spectrometry, nanoparticles, or metal-based anticancer drugs. This study assesses the efficacy of collision/reacton cell (CCT) mode for simultaneously analysing elements across the complete mass spectrum. Furthermore, we demonstrate the accuracy of the LA-ICP-TOFMS methodology through the analysis of serum reference material.

Results

Our findings demonstrate that the CCT mode outperforms the standard/no gas mode for LA-ICP-TOFMS measurements, particularly in quantifying endogenous elements susceptible to interferences. Through the analysis of picolitre-volume micro-droplet standards and serum reference material (deposited as micro-droplets), we observed accurate quantification of elements such as iron and selenium, with isotope ratios closely resembling natural compositions. As key advantage, the utilization of the CCT mode eliminated the need for labor-intensive post-data processing, streamlining analytical procedures. Additionally, the CCT mode also provided enhanced sensitivity (factor of 1.5–2) for elements in the higher mass range compared to the standard mode, without compromising the sensitivity for endogenous elements.

Significance

We successfully integrated Seronorm serum reference material-containing micro-droplets into LA-ICPMS bioimaging workflows for quality control, enabling validated measurements of key biological elements. The use of the CCT mode is highly recommended for bioimaging applications that address the analysis of elements across the entire mass range.

2. Experimental

2.1. Chemicals and reagents

Ultrapure water (18.2 MΩ cm, ELGA Water purification system, Purelab Ultra MK 2, UK) and nitric acid (>69 %, Rotipuran Supra, Carl Roth, Karlsruhe, Germany) were used for dilutions of the standard solutions. A multi-element stock solution and single element standard solutions were purchased from Labkings (Hilversum, The Netherlands). Gelatin was obtained from Sigma-Aldrich (Vienna, Austria). The serum reference material Seronorm (Seronorm™ Trace Elements Serum L-1, Norway) was reconstituted according to the manufacturer's protocol. Solution preparations and LA-ICP-TOFMS measurements were carried out in clean room classes ISO 8 and ISO 7, respectively.

2.2. Calibration standards based on multi-element gelatin micro-droplets

Gelatin-based micro-droplet standards were prepared according to a previously described procedure [20]. For this purpose, liquid multi-element standard solutions were prepared in different concentrations gravimetrically from standard stock solutions and mixed with a fish gelatin solution (10 %, w/w) to reach a final concentration of 1 % (w/w) gelatin. The resulting solutions were transferred into the sample source of a micro-spotter system. A CellenONE X1 micro-spotter (Cellenion, Lyon, France) was used to generate arrays of the gelatin micro-droplet standards onto glass slides with a droplet volume of around 400 pL resulting in droplet sizes of approximately 150–200 μm in diameter. The diameter of the droplets was measured by a built-in camera of the micro-spotting system before and after each spot run and used to calculate the exact volumes of the droplets. The picolitre-volumes were used to establish the absolute elemental amounts within the droplet standards. The slides were stored at room temperature in the dark until LA-ICP-TOFMS analysis and proved to be stable for up to 12 months. The entire micro-droplets were selectively and quantitatively ablated [27] and multi-element analysis was performed by LA-ICP-TOFMS imaging. The multi-element gelatin micro-droplet standards contained 48 elements in different concentrations (summarized in Table S1).

2.3. Dispensing of serum reference material

The serum reference material Seronorm (Seronorm™ Trace Elements Serum L-1, Norway) was reconstituted according to the manufacturer's protocol. The resulting solution was immediately transferred to the sample source of the CellenONE X1 micro-spotter (Cellenion, Lyon, France). Serum reference material was dispensed onto glass slides resulting in micro-droplets with a volume of around 400 pL and a droplet size of around 150–200 μm in diameter. The laser fluence was adjusted to selectively and quantitatively ablate the entire micro-droplets containing the serum reference material without co-ablation of the glass slide. LA-ICP-TOFMS analysis was performed and the elemental concentrations quantified by using the micro-droplet standards with the same laser ablation parameters. The micro-droplets containing the serum Seronorm reference material proved to be stable over 6 months upon storage at room temperature in the dark (refer to Table S2 for stability data).

2.4. LA-ICP-TOFMS measurements

An Iridia 193 nm excimer laser ablation system (Teledyne Photon Machines, Bozeman, MT, USA) was coupled to an icpTOF 2R (TOFWERK AG, Thun, Switzerland) TOF-based ICP-MS instrument. The LA system was equipped with a low-dispersion ablation cell [7,28] within the Cobalt ablation chamber and connected to the ICP-TOFMS system via the aerosol rapid introduction system (ARIS). Through the low-dispersion mixing bulb of the ARIS, an Ar make-up gas flow (∼0.90–1.10 L min⁻¹) was introduced into the He carrier gas flow (0.60 L min⁻¹) before entering the plasma. The LA and ICP-TOFMS settings were optimized on a daily basis while ablating NIST SRM612 glass certified reference material (National Institute for Standards and Technology, Gaithersburg, MD, USA). Optimization was based on high intensities for ²⁶Mg⁺, ⁵⁹Co⁺, ¹¹⁵In⁺ and ²³⁸U⁺, low oxide formation based on the ²³⁸U¹⁶O⁺/²³⁸U⁺ ratio (<2 %) and low elemental fractionation based on the ²³⁸U⁺/²³²Th ⁺ ratio (∼1.1).

Laser ablation sampling was performed in fixed dosage mode 2, at a repetition rate of 250 Hz and using different spot sizes: 20, 10, 5 and 2 μm, resulting in pixel sizes of 10, 5, 2.5 and 1 μm. Selective ablation of the standards and the sample micro-droplets was achieved by selecting an energy density below the ablation threshold of glass and above the ablation threshold of gelatin and the samples [27]. Gelatin micro-droplet standards and Seronorm containing micro-droplets were ablated quantitatively using the same LA parameters and a fluence of 0.40 J cm⁻².

The icpTOF 2R ICP-TOFMS instrument has a specified mass resolution (R = m/Δm) of 6000 (full width half-maximum definition). The standard operation mode was used, which balances mass resolving power, sensitivity and ion transmission across the entire measured mass range and which allows the analysis of ions from m/z = 14–256. The integration and read-out rate match the LA repetition rate. The instrument was equipped with a torch injector of 2.5 mm inner diameter and nickel sample and skimmer cones with a skimmer cone insert of 2.8 mm in diameter. A radio frequency power of 1440 W, an auxiliary Ar gas flow rate of 0.80 L min⁻¹ and a plasma Ar gas flow rate of 14 L min⁻¹ was used. In case the CCT mode was used, the collision/reaction cell was pressurized with a mixture of H₂/He gas with an optimized gas flow rate of 4.20–5.0 mL min⁻¹. Instrumental parameters for LA-ICP-TOFMS measurements are summarized in Table S3.

2.5. Data acquisition and processing

Data was recorded using TofPilot 2.10.3.0 (TOFWERK AG, Thun, Switzerland). The LA-ICP-TOFMS data were saved in the open-source hierarchical data format (HDF5, www.hdfgroup.org). Post-acquisition data processing of data acquired in standard/no gas mode was performed with Tofware v3.2.2.1, which is a TOFWERK data analysis package and used as an add-on on IgorPro (Wavemetrics Inc., Oregon, USA). The data processing comprised the following steps: (1) drift correction of the mass peak position in the spectra over time via time-dependent mass calibration (2) determining the peak shape and (3) fitting and subtracting the mass spectral base-line.

Data from TofPilot (in case the CCT mode was used) and from Tofware (in case the standard mode was used) were further processed with HDIP version 1.8.5. (Teledyne Photon Machines, Bozeman, MT, USA). An integrated script was used to automatically process the files generated by TofPilot or Tofware and to generate 2D elemental distribution maps. For calibration, signal responses for each mass channel monitored during ablation of a single spiked droplet were integrated using HDIP. The sum of the elemental signal intensities and the absolute masses of the respective elements within the droplet standards were used to set up calibration curves. Signal responses for each mass channel monitored during ablation were also integrated for the droplets containing serum reference material, using HDIP.

4. Conclusions

The use of the CCT mode (cell pressurized with a He/H₂ gas mixture) proved to be superior to the standard/no gas mode for LA-ICP-TOFMS bioimaging applications aimed at analyzing elements across the entire mass range. Accurate quantification of endogenous elements that are subjected to interferences was achieved from picolitre-volume micro-droplet standards, as demonstrated by the analysis of micro-droplets containing serum reference material. For iron and selenium, isotope ratios in the CCT mode closely matched the natural ones. In addition, the sensitivity of elements from the higher mass range was better by a factor of 1.5 to 2.0 in the CCT mode compared to the standard mode. In case the CCT mode was used, no laborious post-data strategies were required to obtain accurate results, which is another advantage towards the use of the standard mode.

We have demonstrated the successful integration of Seronorm™ serum reference material into quantitative LA-ICPMS bioimaging workflows as an accuracy control. This validation step of quantitative measurement results can be especially attractive for industry, where quality control is a main requirement. The analysis of n = 3 quality control samples adds around 5 min of analysis time to a calibration sequence providing validated measurements of endogenous elements with biological key functions. Future directions towards one point-calibration approaches can further reduce the time required for calibration and quality control of LA-ICPMS results.