Mass Spectrometry Imaging and Integration with Other Imaging Modalities for Greater Molecular Understanding of Biological Tissues

Abstract

Over the last two decades, mass spectrometry imaging (MSI) has been increasingly employed to investigate the spatial distribution of a wide variety of molecules in complex biological samples. MSI has demonstrated its potential in numerous applications from drug discovery, disease state evaluation through proteomic and/or metabolomic studies. Significant technological and methodological advancements have addressed natural limitations of the techniques, i.e., increased spatial resolution, increased detection sensitivity especially for large molecules, higher throughput analysis and data management. One of the next major evolutions of MSI is linked to the introduction of imaging mass cytometry (IMC). IMC is a multiplexed method for tissue phenotyping, imaging signalling pathway or cell marker assessment, at sub-cellular resolution (1 μm). It uses MSI to simultaneously detect and quantify up to 30 different antibodies within a tissue section. The combination of MSI with other molecular imaging techniques can also provide highly relevant complementary information to explore new scientific fields. Traditionally, classical histology (especially haematoxylin and eosin-stained sections) is overlaid with molecular profiles obtained by MSI. Thus, MSI-based molecular histology provides a snapshot of a tissue microenvironment and enables the correlation of drugs, metabolites, lipids, peptides or proteins with histological/pathological features or tissue substructures. Recently, many examples combining MSI with other imaging modalities such as fluorescence, confocal Raman spectroscopy and MRI have emerged. For instance, brain pathophysiology has been studied using both MRI and MSI, establishing correlations between in and ex vivo molecular imaging techniques. Endogenous metabolite and small peptide modulation were evaluated depending on disease state. Here, we review advanced 'hot topics' in MSI development and explore the combination of MSI with established molecular imaging techniques to improve our understanding of biological and pathophysiological processes.

Keywords: Histology; Imaging mass cytometry (IMC); Magnetic resonance imaging (MRI); Mass spectrometry imaging (MSI); Molecular imaging; Pathology.

Fig. 1

Mass spectrometry imaging (MSI) workflow.

Fig. 2

Comparison of main ionisation techniques employed for MSI.

Fig. 3

Gastric cancer TMA sample analysed by MSI showing the distribution of five native glycan fragments (HexNAc-HexNAc, HexNAcS, HexA-HexAc, HexS and Sia-hex). These glycans are specific for tissue compartments according to histological overview from H&E image and patients’ prognosis. Reproduced from [13].

Fig. 4

(a, upper) NanoSIMS image of the ¹³C¹⁴N⁻/¹²C¹⁴N⁻ ratio image reveals the dopamine enrichment in single vesicles in pheochromocytoma (PC12) cells. Red arrows highlight three examples of vesicles. Reproduced from [34]. (a, lower) NanoSIMS image of ¹²C¹⁵N⁻/¹²C¹⁴N⁻ ratio showing low incorporation of new proteins in stereocilia after feeding mice for 56 days with food containing ¹⁵N-leucine. Reproduced from [35]. (b, upper) ToF-SIMS images of NR8383 macrophage doped with desethylamiodarone (DEA) in negative ion. Snapshot images acquired using the Bi₃⁺ ion beam for analysis and a GCIB for sample etching at different stages of the 3D image acquisition iodine, I⁻ (green), and the summed ion contribution of the nuclear-markers (red). (b, lower) 3D isosurface rendering of the doped cell. I⁻ is mapped in green and nuclear marker, HP₂O₆⁻, is mapped in red. Reproduced from [36]. (c) ToF-SIMS images of a human breast cancer biopsy using a (CO₂)_6k⁺ GCIB with H&E-stained image from a consecutive tissue slice. Three different phosphatidylinositol (PI) lipids distribute differently between the tumour and the inflammatory cells in the surrounding stroma. Reproduced from [37].

Fig. 5

Imaging mass cytometry (IMC) workflow. Reproduced from [51].

Fig. 6

Selection of IMC images from various applications adapted from [53, 54]. (a) Tissue architecture: representative total ion current image (left) of mouse abdominal wall, overlay of three tissue type markers (middle) β-actin, histone H3 and iridium DNA intercalator, (right) E-cadherin, collagen and αSMA. (b) Functional markers: representative IdU, ¹⁹⁵Pt and histone H3 images of control and cisplatin-dosed (4 mg/kg for 24 and 48 h) small mouse intestine. (c) Tumour microenvironment (TME): pancreatic cancer patient–derived xenograft (PDX) assessment using EF5 tracer, E-cadherin and Ki-67. (d) Target engagement: control and dosed pancreatic cancer PDX tissue evaluation.

Fig. 7

Example of a multimodal study combining MRI, 3D-MALDI-MSI and histology (H&E). (a) Semi-transparent MRI volume rendering overlaid with the semi-transparent clusters from 3D-MALDI-MSI data showing the renal cortex (blue), the medulla (green) and pelvis (red). (b) Overlay of the 3D molecular map of ion species at m/z 4808 with MRI volume rendering. (c) Overlay of H&E-stained section from the inner kidney with 3D segmentation map of the same tissue. Reproduced from [82].