Transmission-type atmospheric-pressure laser/photochemical ionisation mass spectrometry imaging source (t-AP-LDI/PI) for in situ imaging of metabolites in biological tissues

Areas of application: Life sciences; neurobiology; pathological diagnosis;

‌Introduction

Mass spectrometry imaging (MSI) is a core analytical technique for the label-free, in situ analysis of the spatial distribution of metabolites, lipids and proteins within biological tissues. Atmospheric pressure open-path mass spectrometry imaging has attracted widespread attention due to its advantages of simple sample preparation and in situ, non-destructive detection. Traditional laser desorption/ionisation (LDI) suffers from extremely low ion yield and insufficient sensitivity. The transmission-type ambient-pressure laser/photochemical ionisation mass spectrometry imaging source (t-AP-LDI/PI) employed in this experiment utilises krypton lamp vacuum ultraviolet (VUV) soft ionisation technology, coupled with a proprietary bubble-doped gas supply system, to significantly enhance molecular ionisation efficiency, enabling high-resolution spatial imaging of various metabolites and lipids in brain tissue and clinical melanoma samples without the need for a matrix, thereby providing a new in situ analytical tool for research into neurobiology and the metabolism of the tumour microenvironment.

‌Materials and Methods

Instrumentation: transmission-type atmospheric-pressure laser/photochemical ionisation mass spectrometry imaging source (t-AP-LDI/PI), coupled with an Agilent 6224 high-resolution time-of-flight mass spectrometer (resolution 20,000).

Reagents and carrier gases: chromatographic-grade toluene, methanol and pure water; high-purity nitrogen (99.999 per cent) as the carrier gas and doped transport gas.

Biological samples:

Mouse brain tissue: cerebellar and hippocampal tissue flash-frozen in liquid nitrogen, with frozen sections 10 μm thick.

Human melanoma tissue: Surgical specimen of a skin tumour on the thumb, frozen in liquid nitrogen at –80 °C; 10 μm frozen sections, with corresponding H&E-stained pathological sections as controls.

Figure 1  Schematic diagram of the t-AP-LDI/PI MSI experimental setup

‌Results and Conclusions

1. Doping-assisted ionisation significantly enhances detection sensitivity

Transmission atmospheric pressure laser desorption (t-AP-LDI) alone can only detect mass spectrometry signals of extremely low abundance; however, enabling direct photoionisation via a krypton lamp allows for the detection of a wider range of small molecules; the introduction of toluene as a dopant induces proton transfer and charge exchange, the abundance of metabolites and lipid ions increases by 1 to 3 orders of magnitude, enabling the simultaneous detection of over a hundred endogenous metabolites and lipids in mouse brain tissue, covering a wide range of substances including amino acids, neurotransmitters, glycerides and phospholipids. This detection coverage surpasses that of mainstream ambient-pressure imaging techniques such as DESI/PI, MALDI and AP-MALDI.

Figure 2 (A) Relationship between the distance between the slide and the delivery tube and the ionic strength of five compounds. (B–D) Background-subtracted mass spectra of mouse brain homogenate under t-AP-LDI, t-AP-LDI/PI without dopant, and t-AP-LDI/P1 with dopant. (E) Histograms of the fold changes in representative metabolites and lipids in mouse brain homogenates, obtained via t-AP-LDI/PI (with additive)/t-AP-LDI/PI (without additive) and t-AP-LDI/PI (without additive)/t-AP-LDI. (F) Species classification Venn diagram for t-AP-LDI/PI, DESI/PI, MALDI and AP-MALDI-MS in positive ion mode.

2. Metabolic imaging of mouse cerebellar tissue with 10 μm resolution

In both positive and negative ion modes, the molecular distribution characteristics of the cerebellar molecular layer (ML), granule layer (GL) and white matter (WM) subregions can be clearly distinguished:

Granular layer (GL): enriched in histamine, adenine, guanine and aspartic acid, associated with neuronal nucleic acids and nerve signal transmission;

Molecular layer (ML): concentrated distribution of lipids such as diglycerides (DG);

White matter (WM): Highly enriched in cholesterol and vitamin D3; this is the core region for steroid metabolism in the cell membrane; the superimposition of molecular ion images from multiple categories provides a visual representation of the complementary metabolic distribution patterns across the three layers.

 Figure 3: Average mass spectrometry and t-AP-LDI/PI mass spectrometry imaging of the mouse cerebellum in positive and negative ion modes. (T, O) Optical images of the cerebellum. ML, molecular layer; GL, granule layer; WM, white matter. The stage step size was set to 10 μm. Scale bar: 500 μm.

3. Achieving 4 μm ultra-high spatial resolution imaging of mouse hippocampal tissue

At a pixel resolution of 4 μm, fine subregions of the hippocampal CA1 zone, dentate gyrus (DG) and corpus callosum (CC) can be distinguished: adenine is concentrated in the CA1 and dentate gyrus, which are rich in neuronal cell bodies; cholesterol is distributed in a punctate pattern within the corpus callosum, enabling the visualisation of metabolic differences in subregions of brain tissue at the single-cell level.

Figure 4 (C) Optical image and (D–O) MS images of mouse hippocampal tissue sections obtained using t-AP-LDI/PI. Figure (P) shows distinct differences between subregions, including CA1, the dentate gyrus (DG), the corpus callosum (CC) and the superior colliculus (BSC). The solid yellow lines in the optical image indicate MSI regions; the scale bar is 100 μm.

4. Clinical tissue analysis of tumour metabolic heterogeneity in human melanoma

Layered imaging of skin melanoma sections clearly distinguishes four major regions: the stratum corneum, the granular layer, the tumour lesion and normal tissue:

Stratum corneum: High expression of moisturising amino acids such as N-acetylamide, GABA and aspartic acid;

Stratum granulosum: Characteristic enrichment of adenosine and long-chain diglycerides;

Dense tumour foci: significant upregulation of fatty acids such as cholesterol, vitamin D3, palmitic acid and oleic acid, suggesting abnormal melanocyte proliferation;

Metabolic pathways: The pathways for triglyceride and phospholipid synthesis are activated within the tumour, whilst phospholipid molecules associated with the PI3K/AKT invasion-promoting pathway accumulate in large quantities. This clearly reveals the metabolic heterogeneity of the tumour microenvironment and provides a basis for the screening of pathological molecular biomarkers for melanoma.

Figure 5  MS images of representative compounds in melanoma obtained using t-AP-LDI/PI in positive and negative ion modes. (A) Optical and H&E-stained images of a melanoma tissue section, along with H&E-stained images at 72.8× magnification of different regions of the melanoma tissue. Solid black, green, red and blue lines represent the stratum corneum, stratum granulosum, tumour tissue (areas of dense melanoma cells) and normal tissue, respectively. (B–K) Ion images with characteristic distributions selected in cation mode. Ion images with characteristic distributions (M–P, R–U) selected in anion mode.

Conclusions

The transmission-type ambient-pressure laser/photochemical ionisation mass spectrometry imaging source (t-AP-LDI/PI), coupled with a stable bubbling-doped gas supply system, overcomes the shortcoming of low sensitivity in ambient-pressure laser ionisation, enhancing the ion signal by 1 to 3 orders of magnitude. The complete system is highly versatile and can be adapted to various commercial mass spectrometer sample ports.

This method requires no complex sample preparation and enables imaging of routine tissue sections at 10 μm resolution and ultra-high-resolution imaging of micro-regions in brain tissue at 4 μm resolution. It has been successfully applied to mouse neural tissue and human clinical melanoma sections, visualising the spatial distribution of amino acids, neurotransmitters, fatty acids and various lipids, and providing an intuitive analysis of metabolic patterns in different brain regions and metabolic heterogeneity within the tumour microenvironment.

‌References

[1] Qi, Keke; Lv, Yongmei; Xiong, Ying; Tian, Changlin*; Liu, Chengyuan*; Pan, Yang*, Development of Transmission Ambient Pressure Laser Desorption Ionisation/Post-photoionisation Mass Spectrometry Imaging (t-AP-LDI/PI MSI), Analytical Chemistry, 2024, accepted‌