Analysis of Citrate Distribution in Plant Tissues Using Multimodal Imaging

Multimodal Imaging of Citrate Distribution in Strawberry Fruit: Combining MALDI-MSI, LA-ICP-MS and HPLC Quantification

Importance of the topic

High-resolution spatial mapping of small organic acids in plant tissues is central to studies in plant physiology, food science and quality control. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI‑MSI) provides detailed molecular images but often detects small organic acids as metal-adduct ions (e.g., [M+K]+). Because metal ions such as potassium are heterogeneously distributed in plant tissues, validating that adduct-based MSI images reflect true analyte distribution (and not simply metal heterogeneity) is essential. Multimodal workflows that combine molecular imaging, elemental mapping, and orthogonal quantitative analysis enable robust interpretation and increase confidence in biological conclusions.

Objectives and study overview

This study evaluated whether heterogeneous potassium distribution in strawberry fruit tissue biases MALDI‑MSI images of citric acid detected as [M+K]+. The approach paired positive‑ion MALDI‑MSI (iMScope QT) with laser ablation inductively coupled plasma mass spectrometry imaging (LA‑ICP‑MS) to map potassium on the same tissue section, and used a dedicated organic acid HPLC system (Nexera Organic Acid Analysis System) to quantify citrate in adjacent tissue punches for validation and correlation with MSI data.

Methodology and sample preparation

Key experimental features:

• Biological material: Fragaria × ananassa ‘Tochiotome’ frozen fruit; cryosections 90 µm mounted on ITO‑coated slides.
• MALDI sample prep: 2,5‑dihydroxybenzoic acid (DHB) matrix applied by automated spray (iMLayer AERO), measurement in positive‑ion mode to target m/z 190–250; spatial pitch ≈50 µm; citrate observed as potassium adduct [M+K]+.
• MALDI instrumentation: iMScope QT coupled with LCMS‑9050 mass spectrometer; MSI data processed in IMAGEREVEAL MS software.
• LA‑ICP‑MS: same section analyzed after MALDI to map elemental K (m/z 39) using an imageBIO266 laser ablation system coupled to an ICPMS‑2050; spot/scan geometry chosen for imaging compatible with MALDI spatial resolution.
• Quantification (HPLC): twelve tissue pieces adjacent to the imaged section (≈2×4×0.56 mm) were excised, freeze‑dried, homogenized, extracted in water (70 °C heating), centrifuged and analyzed using the Nexera Organic Acid Analysis System with conductivity detection and Shim‑pack SCR‑102H columns. External calibration (2.5–50 mg/L) provided quantitative citrate concentrations.

Used instrumentation

• MALDI imaging: iMScope QT with LCMS‑9050 mass spectrometer; iMLayer AERO automatic matrix sprayer.
• Elemental imaging: imageBIO266 laser ablation system coupled to ICPMS‑2050 (LA‑ICP‑MS).
• Quantitative organic acid analysis: Nexera Organic Acid Analysis System with Shim‑pack SCR‑102H analytical and guard columns; conductivity detection.
• Data analysis: IMAGEREVEAL MS for MSI and LA‑ICP‑MS data integration and visualization.

Main results and discussion

Summary of key findings:

• MALDI‑MSI (positive mode) visualized citric acid as its potassium adduct with the highest ion intensities in the outer cortical region (calyx side) and lower intensities toward the pith.
• LA‑ICP‑MS mapped potassium distribution on the same section and showed potassium concentrated in the outer cortex and vascular bundles. However, the potassium map did not spatially match the citrate [M+K]+ distribution from MALDI‑MSI.
• HPLC quantification of citrate in the twelve adjacent tissue pieces produced a concentration gradient with highest citrate in the outermost cortex decreasing inward, matching the MALDI‑MSI trend.
• Regression of MALDI mean ion intensities against HPLC‑quantified citrate showed strong linear correlation (R2 > 0.96), demonstrating that MALDI‑MSI signal intensity reflected true citrate concentration across regions despite detection as a K‑adduct.

Interpretation:

Although citric acid is observed as [M+K]+ in positive‑ion MALDI‑MSI, the MSI distribution is not an artifact of local potassium heterogeneity under the experimental conditions used. The strong quantitative correlation to HPLC validates MALDI‑MSI as a reliable indicator of regional citrate concentration in this plant system. Differences in the spatial distribution of elemental potassium and citrate adduct intensity suggest that formation of the potassium adduct in situ is not strictly limited by local bulk potassium concentration, likely influenced by local microchemistry, matrix effects and ionization efficiencies.

Benefits and practical applications of the method

• Provides a validated multimodal workflow to distinguish true molecular distributions from potential adduct‑related artifacts in MSI studies.
• Enables simultaneous investigation of molecular and elemental spatial patterns on the same tissue section, improving biological interpretation (e.g., nutrient transport, metabolite localization).
• Useful for plant physiology research, breeding, postharvest quality assessment and food science where spatial metabolite information is critical.
• Combining MSI with orthogonal quantitative HPLC increases confidence for semi‑quantitative or quantitative imaging applications.

Future trends and applications

• Advances in quantitative MSI: development of better internal standards, normalization strategies and calibration protocols to move from relative to absolute concentration maps.
• Improved multimodal co‑registration: automated spatial alignment between molecular and elemental images for more rigorous pixelwise comparisons.
• Extension to other metabolites and adduct types: systematic evaluation of different adducting ions and ionization modes (positive/negative) across plant tissues.
• Integration with isotopic labeling, high‑throughput sectioning and machine learning for large‑scale spatial metabolomics in crops and foods.
• Enhanced software interoperability and standardized data formats to streamline combined analysis using tools such as IMAGEREVEAL MS.

Conclusion

The combined imaging approach (MALDI‑MSI + LA‑ICP‑MS) with independent HPLC quantification demonstrates that positive‑ion MALDI imaging of citric acid as [M+K]+ accurately represents the spatial concentration distribution in strawberry fruit tissue under the presented conditions. Multimodal validation strengthens confidence in MSI results and supports broader application of adduct‑based MSI for spatial metabolite studies in plant and food science.

References

• Enomoto, K.; et al. J. Agric. Food Chem. 2018, 66, 4958–4965.