Advancements in ToF-SIMS imaging for life sciences

Abstract

In the last 2 decades, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) has gained significant prominence as a powerful imaging technique in the field of life sciences. This comprehensive review provides an in-depth overview of recent advancements in ToF-SIMS instrument technology and its applications in metabolomics, lipidomics, and single-cell analysis. We highlight the use of ToF-SIMS imaging for studying lipid distribution, composition, and interactions in cells and tissues, and discuss its application in metabolomics, including the analysis of metabolic pathways. Furthermore, we review recent progress in single-cell analysis using ToF-SIMS, focusing on sample preparation techniques, in situ investigation for subcellular distribution of drugs, and interactions between drug molecules and biological targets. The high spatial resolution and potential for multimodal analysis of ToF-SIMS make it a promising tool for unraveling the complex molecular landscape of biological systems. We also discuss future prospects and potential advancements of ToF-SIMS in the research of life sciences, with the expectation of a significant impact in the field.

Keywords: ToF-SIMS; life science; lipidomics; metabolomics; single cell imaging.

FIGURE 1

A schematic diagram illustrating the fundamental principles of ToF-SIMS.

FIGURE 2

Imaging principal component analysis (PCA) images of the mouse brain cerebellum using four different cluster ion beams: 20 keV (H₂O)₆₀₀₀ ⁺, 20keV (H₂O)Ar₂₀₀₀ ⁺, 20 keV Ar₂₀₀₀ ⁺ and 20 keV C₆₀ ⁺. These images show the clear separation between the grey and white matter of the brain. PCA analysis using MATLAB yielded eight principal components (PCs) that capture most of the spectral variance within the dataset. The PCA results are visualized as color density plots representing the scores from each PC. Green pixels indicate positive loadings, while red pixels represent negative loadings. The PC loadings plots confirm that one of the main contributors to the variance between white and grey matter is the cholesterol [M+H−H₂O]⁺ ion at m/z 369. The area covered by each image is 4 mm × 4 mm with a total ion dose of 1 × 10¹² ions cm⁻². (Berrueta Razo et al., 2015).

FIGURE 3

Schematic of the HybridSIMS spectrometer with ToF and Orbitrap mass analyzer (Passarelli et al., 2017).

FIGURE 4

Identification of unique molecular ions of purine nucleotides in the intracellular pool by in situ GCIB-SIMS. (A) Schematic of GCIB-SIMS imaging of HeLa cells. Imaging uses a finely focused 70 keV (CO₂)_n ⁺ (n > 10,000) cluster beam to interrogate frozen hydrated HeLa cells three-dimensionally at 1-μm spatial resolution. Coupled with a buncher-ToF and direct-current beam setup, maximum spatial resolution and mass resolution can be retained. A pixel-by-pixel analysis was performed across a lateral field of view of 256 mm × 256 mm. (B) Mass spectra in the m/z range 0–900 were recorded for each pixel. (C) A composite two-dimensional colored image was generated combining the signal across all the layers PI (38:4; green) at m/z 886.53, phosphate-sugar backbone at m/z 257.10 (blue) from nucleotides, and ¹⁵N-enriched DNPB intermediate AICAR (red). Combination of mass spectral analysis and the spatial distribution of specific cellular signals demonstrates the reliability of the method for in situ biochemical studies (Pareek et al., 2020).

FIGURE 5

ToF-SIMS images display the distribution of lipid headgroup fragments in fly brain sections of control and modafinil treated flies imaged by ToF-SIMS. (A,B) Ion images of the PC and SM head group at m/z 184.1 in the positive ion mode; (C,D) PC fragment at m/z 224.1 in the positive ion mode; and (E–H) PE fragments at m/z 140.0 and 180.1 in the negative ion mode. All the images were recorded with the ToF-SIMS V instrument equipped with 25 keV Bi₃ ⁺⁺ as a primary ion beam. The primary ion beam current was 0.3 pA and the total ion dose was 2 × 10¹² ions cm⁻² (Philipsen et al., 2021).

FIGURE 6

Z-corrected 3D images of BrdU localized within cells using two different LMIG operating modes. The bottom left overlay is obtained from the high mass resolution bunched mode with the BrdU⁻ signal(⁸¹Br⁻, m/z 81) shown in blue and the sum of the C_xH_yO_z ⁻ signals(C₂HO⁻, m/z 41, C₂H₃O⁻, m/z 43, CHO₂ ⁻, m/z 45, C₃H₃O⁻, m/z 55, C₂H₂O₂ ⁻, m/z 58, C₂H₃O₂ ⁻, m/z 59, C₃HO₂ ⁻, m/z 69, C₃H₃O₂ ⁻, m/z 71) in red. The other three images are burst mode images. The top left: CN⁻(m/z 26) + CNO⁻(m/z 42), shows the location and shape of each cell; top right: ΣC_xH_yO_z ⁻; bottom right: ΣBrdU⁻(⁸¹Br⁻, m/z 81, C₄H₂N₂O₂ ⁷⁹Br⁻, m/z 189, C₄H₂N₂O₂ ⁸¹Br⁻, m/z 191). The bottom left image is 202 × 202 μm² and contains 24 slices. The burst mode images are 165 × 165 μm² and contain 26 slices (Brison et al., 2013).

FIGURE 7

Schematic diagram of visualizing proteins in single cells by ToF-SIMS coupled to genetically encoded chemical tags (Jia et al., 2020). The upper image was generated from the signal of F⁻ (represent protein, green) at m/z 19, and signal of PO₃ ⁻ (represent the cell, red) at m/z 79, while in the bottom figures, the signal of [Pt(CN)]⁻(red) at m/z 221 represents cisplatin.