Semiquantitative Analysis for High-Speed Mapping Applications of Biological Samples Using LA-ICP-TOFMS

Abstract

Laser ablation (LA) in combination with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) enables monitoring of elements from the entire mass range for every pixel, regardless of the isotopes of interest for a certain application. This provides nontargeted multi-element (bio-)imaging capabilities and the unique possibility to screen for elements that were initially not expected in the sample. Quantification of a large range of elements is limited as the preparation of highly multiplexed calibration standards for bioimaging applications by LA-ICP-(TOF)MS is challenging. In this study, we have developed a workflow for semiquantitative analysis by LA-ICP-TOFMS based on multi-element gelatin micro-droplet standards. The presented approach is intended for the mapping of biological samples due to the requirement of matrix-matched standards for accurate quantification in LA-ICPMS, a prerequisite that is given by the use of gelatin-based standards. A library of response factors was constructed based on 72 elements for the semiquantitative calculations. The presented method was evaluated in two stages: (i) on gelatin samples with known elemental concentrations and (ii) on real-world samples that included prime examples of bioimaging (mouse spleen and tumor tissue). The developed semiquantification approach was based on 10 elements as calibration standards and provided the determination of 136 nuclides of 63 elements, with errors below 25%, and for half of the nuclides, below 10%. A web application for quantification and semiquantification of LA-ICP(-TOF)MS data was developed, and a detailed description is presented to easily allow others to use the presented method.

Figure 1

Sum of the squares of residuals in the plot shows the results obtained from 1 million iteration bootstrapping procedures for different numbers of elements used for the semiquantitative approach.

Figure 2

Heatmaps of the percentage deviations from the true value for quantitative and semiquantitative calibrations. Each semiquantitative calibration heatmap was made using the selection of elements resulting in the lowest RSS. Every square represents an average of three replicates.

Figure 3

Concentration maps of (A) ³¹P⁺ and (B) ⁵⁶Fe⁺ in a mouse tumor section, and of (C) ³¹P⁺ and (D) ⁵⁶Fe⁺ in a mouse spleen section, determined by the semiquantitative method and LA-ICP-TOFMS analysis.

Figure 4

Elemental maps of a murine tumor section representing the quantitative maps of ¹⁵³Eu and ⁶³Cu. On the left part, the elemental maps based on the quantitative approach, and on the right part, the elemental maps based on the semiquantitative approach are shown.