Mass Spectrometry Imaging Technology Accelerates Clinical Translation: A New Tool for Molecular Pathological Diagnosis

Mass spectrometry imaging technology is moving from research laboratories into clinical practice. At several recent academic conferences, advances in mass spectrometry imaging in areas such as tumour grading, the discovery of disease biomarkers and the tracking of drug metabolism have been the subject of lively discussion. As the detection capabilities and widespread adoption of mass spectrometry imaging systems continue to improve, molecular pathology diagnosis is set to benefit from a entirely new tool for spatial analysis.

Clinical needs: Information gaps in traditional pathological techniques

Pathological diagnosis is the cornerstone of clinical medicine. Currently, histopathological diagnosis relies primarily on H&E staining to observe tissue morphology, immunohistochemistry (IHC) to detect the expression of specific proteins, and fluorescence in situ hybridisation (FISH) to detect genetic abnormalities. Whilst these techniques each have their own focus, they share common limitations: some provide only morphological information without data on molecular composition (H&E); others target predefined markers and cannot perform non-targeted detection (IHC/FISH); whilst others provide molecular information but lack spatial localisation (PCR/sequencing).

In clinical pathological diagnosis, information such as the metabolic heterogeneity of tumour tissue, the precise localisation of tumour boundaries, and the spatial expression patterns of hormone receptors is crucial for the formulation of treatment plans; however, traditional techniques struggle to simultaneously obtain comprehensive information regarding ‘which molecules are present’ and ‘where they are located’.

Mass spectrometry imaging systems scan tissue sections point by point to simultaneously capture the spatial distribution characteristics of hundreds of molecules. Offering the advantages of being free from fluorescent labelling and complex pre-processing, they effectively bridge this information gap.

Technological Evolution of Mass Spectrometry Imaging Ion Sources

The clinical application potential of mass spectrometry imagers depends to a large extent on the detection capabilities of their ion sources. The technical bottlenecks faced by traditional mass spectrometry imaging sources directly impact the depth of clinical application.

Taking the DESI imaging source as an example, its electrospray mechanism can only effectively ionise highly polar molecules; non-polar molecules such as cholesterol, triglycerides and S1P are virtually undetectable. However, cholesterol metabolism abnormalities are characteristic of many tumours, and S1P is a key signalling molecule in tumour angiogenesis and invasion—these non-polar molecules, which are of great significance in tumour diagnosis, are precisely the detection blind spots of traditional DESI mass spectrometry imaging ion sources.

In 2024, the research group led by Professor Pan Yang at the University of Science and Technology of China published a cover article in *Analytical Chemistry*, in which they developed a transmission-type laser desorption/post-photoionisation (t-AP-LDI/PI) mass spectrometry imaging method. By incorporating a vacuum ultraviolet photochemical ionisation device following laser desorption to perform secondary ionisation of neutral molecules, this method enhances the detection sensitivity of various metabolites—including histamine, choline, cholesterol, glutamate and ceramides—by 2–3 orders of magnitude. In imaging melanoma tissue, the spatial distribution differences in metabolites between tumour regions and normal tissue showed a high degree of correlation with H&E histological sections, revealing the metabolic heterogeneity of the tumour microenvironment.

This technological achievement has been commercialised by Hefei Xinceyuan Instrument Technology Co., Ltd. as the MSI LDPI laser/photochemical ionisation mass spectrometry imaging source, which achieves a spatial resolution of 2–3 μm and can resolve the molecular boundaries between tumour and normal tissues.

The pathway from research to clinical application

Mass spectrometry imagers face several key challenges in clinical translation, and the development of domestically produced mass spectrometry imaging sources is addressing these one by one.

Detection coverage. Key molecules in clinical pathology samples fall into three major categories: polar, weakly polar and non-polar. Hefei Xinceyuan’s MSI DPI electrospray/photochemical ionisation mass spectrometry imaging source, utilising a dual-ionisation architecture combining DESI and photochemical secondary ionisation, eliminates polarity bias and can detect hundreds of lipids, amino acids and secondary metabolites in both positive and negative ion modes. In studies of melanocytic naevi, the spatial distribution of markers such as S1P, cholesterol and PC34:1 showed a high degree of correlation with H&E staining, providing spatial marker information for molecular pathological diagnosis.

Sample integrity. Clinical samples are precious, and the ability to proceed with conventional pathological testing after imaging is a key consideration. Neither the MSI DPI nor the MSI LDPI mass spectrometry imaging sources require matrix spraying; tissue sections remain intact after imaging and can undergo H&E staining and immunohistochemical testing. This means that mass spectrometry imaging and conventional histopathological diagnosis can be performed sequentially on the same section, allowing molecular and morphological information to be complementarily integrated.

Quantitative accuracy. Clinical diagnostics demand a high degree of reliability in quantitative analysis. The ion suppression effect of traditional DESI imaging sources leads to inaccurate quantification of weakly polar components in complex samples. The post-photoionisation mode utilises the direct interaction between photons and neutral molecules, without relying on charge competition; this significantly reduces the ion suppression effect, thereby improving the accuracy of quantitative analysis of pathological biomarkers.

Equipment accessibility. Clinical pathology departments have limited equipment budgets, and the barrier to purchasing a complete mass spectrometry imaging system is high. The MSI DPI mass spectrometry imaging source is compatible with the three major mass spectrometry brands—Agilent, AB SCIEX and Thermo. Pathology laboratories with existing mass spectrometers can simply retrofit the imaging source to gain mass spectrometry imaging capability, thereby lowering the barrier to equipment investment.

Application Validation: From Tumours to Neurodegenerative Diseases

Case studies demonstrating the application of mass spectrometry imaging in clinical research are accumulating.

In the field of cancer research, the MSI LDPI mass spectrometry imaging source analysed the metabolic heterogeneity of melanoma tissue at a resolution of 2–3 μm, clearly distinguishing differences in metabolite distribution between the stratum corneum, stratum granulosum, tumour regions and normal tissue. Metabolic reprogramming in tumours is considered a new marker of malignant transformation across various types of cancer; mass spectrometry imagers can simultaneously capture the spatial distribution of multiple metabolites within tumour tissue, providing valuable insights into the molecular mechanisms underlying tumourigenesis.

In the field of neuroscience research, the MSI DPI mass spectrometry imaging source, when used for imaging mouse brain sections in DESI/PI mode, has enabled the detection of neurotransmitters such as GABA, glutamate and adenosine, as well as non-polar molecules including cholesterol and GalCer lipids. GABA is the brain’s primary inhibitory neurotransmitter, whilst glutamate is the most abundant excitatory neurotransmitter—these molecules are entirely invisible in conventional DESI, yet are crucial for research into neurodegenerative diseases. In 4 μm resolution imaging of mouse hippocampal tissue, differences in metabolite distribution across subregions such as the dentate gyrus, the arm of the superior colliculus and the hippocampal cornua are clearly discernible.

In terms of pharmacokinetics, mass spectrometry imaging extends PK studies from the temporal to the spatial dimension, enabling the tracking of drug and metabolite distribution across different organs and tissue regions. The DESI/PI mode of the MSI DPI compensates for the detection blind spots of conventional DESI in pharmaceutical analysis by enhancing the detection capability of weakly polar and non-polar drug molecules (such as steroid drugs and fat-soluble vitamins).

Drivers of Industry Development

The transition of mass spectrometry imaging to clinical practice is driven by several factors.

From a technical perspective, the advent of post-photoionisation mass spectrometry imaging ion sources has resolved issues of polarity discrimination and low sensitivity, enabling mass spectrometry imagers to meet the fundamental requirements for clinical applications in terms of detection range and detection limits. The launch of Hefei Xinceyuan’s two products—the MSI DPI and MSI LDPI—has provided domestic clinical laboratories with domestically manufactured equipment options.

At the academic level, a series of findings published by Professor Pan Yang’s research group in journals such as *Analytical Chemistry* and *Talanta* have provided academic validation for the application of mass spectrometry imaging in pathological diagnosis. Research conducted by institutions such as the China Academy of Chinese Medical Sciences, utilising DESI/PI mass spectrometry imaging to elucidate the aetiology of Atractylodes maculata with cinnabar spots (published in *Molecular Horticulture*), has demonstrated the application potential of mass spectrometry imaging in the field of traditional Chinese medicine.

At the industrial level, the compatibility design and localised services of domestically produced mass spectrometry imaging sources have lowered the barriers to entry and maintenance costs for clinical laboratories, thereby creating the conditions for the widespread clinical adoption of mass spectrometry imaging technology.

Challenges and Outlook

The clinical translation of mass spectrometry imaging instruments still faces challenges. A standardised molecular pathology atlas database has yet to be established, and the accumulation of a large number of clinical samples is required to define standard molecular spatial distribution characteristics for different diseases. The integration of AI-assisted diagnostic algorithms with mass spectrometry imaging data remains in its early stages, and the ability to automatically recognise disease-specific patterns needs to be improved. There remains a gap between the imaging speed required for rapid intraoperative diagnosis and the current scanning speed of mass spectrometry imaging.

Nevertheless, the potential of mass spectrometry imaging technology in the field of molecular pathology diagnosis has been validated. With the continuous improvement in the detection capabilities of domestically produced mass spectrometry imaging ion sources and the gradual increase in the prevalence of such equipment, mass spectrometry imaging systems are expected to evolve from research tools into one of the standard tools for clinical molecular pathology diagnosis.

Hefei Xinceyuan has stated that the company will continue to advance the validation and optimisation of mass spectrometry imaging sources in clinical settings, whilst conducting collaborative research with medical institutions and pathology diagnostic centres to facilitate the clinical translation and implementation of mass spectrometry imaging technology.