Mass Spectrometry Imaging (MSI) is a technique that acquires mass spectra point by point across the sample surface according to a predefined raster, without destroying the sample's spatial position information, and maps the signal intensity of specific mass-to-charge ratio (m/z) ions into two- or three-dimensional pseudo-color images. Its core feature is 'simultaneously obtaining molecular identity and spatial coordinates', enabling researchers to observe the distribution differences of hundreds to thousands of molecules in situ within tissue.
Unlike methods such as immunohistochemistry (IHC) and immunofluorescence that require pre-selecting targets and applying labels, MSI is a 'discovery-type' technique: it can simultaneously present the in-situ distribution of multiple molecules without prior hypotheses, making it especially suitable for unknown biomarker screening, metabolic heterogeneity research, and tracking the spatial distribution of drugs. For this reason, it often complements transcriptomics and proteomics within the spatial omics framework.
A complete mass spectrometry imaging workflow typically consists of four steps. Sample preparation: usually frozen or paraffin-embedded tissue sections mounted on conductive microscope slides; MALDI routes additionally require matrix spraying, while ambient matrix-free routes such as DESI/LDPI can omit this step. Raster scanning ionization: the imaging ion source desorbs/ionizes the sample surface point by point at a set step size (pixel spacing), which directly determines the spatial resolution. Mass analysis: ions generated at each ionization enter the mass analyzer (commonly TOF, Orbitrap, Q-TOF) to obtain the full mass spectrum for that pixel. Image reconstruction: software colors the intensity at the selected m/z by coordinates to generate ion images, and can overlay multiple images for co-localization analysis.
Among these steps, the coupling of 'ionization' and 'imaging' is what distinguishes MSI from conventional mass spectrometry — conventional MS is concerned with 'what is in this sample', whereas MSI is concerned with 'what is at each position of this sample, and how much'. Therefore, the imaging ion source (rather than a general-purpose ion source) is the hardware core of MSI. The specific ion source route significantly affects resolution, sample preparation, and applicable molecules; see the comparison below.
The three most widely used imaging ion sources in research and clinical practice are MALDI, DESI, and SIMS, which differ markedly in ionization environment, whether matrix is required, resolution scale, and the molecules they excel at. MALDI mostly works under vacuum and requires matrix spraying, with resolution typically on the order of tens of micrometers, and excels at proteins, peptides, lipids, and sugars; DESI is ambient open-air and matrix-free, with resolution mostly from tens to hundreds of micrometers, and excels at metabolites, lipids, and drug distribution; SIMS bombards the sample with primary ions under vacuum, achieving sub-micron to nanometer resolution, but it is a hard ionization method with many fragments, better suited for elements, small molecules, and fragment distribution.
In addition to the three mainstream sources, there are derivative or improved routes such as LDPI, DPI, AP-SMALDI, nano-DESI, LAESI, and AFADESI, which optimize matrix-free operation, ambient conditions, sensitivity, or throughput along different dimensions. Choosing which route is essentially a trade-off among 'resolution, sample preparation complexity, molecular coverage, and whether vacuum is required'.
| Ion source | Ionization environment | Matrix required | Typical resolution scale | Molecules excelled at |
|---|---|---|---|---|
| MALDI | Vacuum (ambient versions exist) | Required | Tens of micrometers | Proteins, peptides, lipids, sugars |
| DESI | Ambient open-air | Not required | Tens to hundreds of micrometers | Small molecules, lipids, metabolites, drugs |
| SIMS / TOF-SIMS | High vacuum | Not required | Sub-micron to nanometer | Elements, small molecules, lipids (fragments) |
| LDPI (Neo-Source) | Ambient | Not required (matrix-free) | 2–3 μm | Metabolites, drugs, lipids (low background for small molecules) |
The advantages of MSI fall into four areas: label-free — most routes do not rely on specific antibodies or fluorescent probes, reducing inter-batch variation; multi-molecule parallelism — a single experiment can cover multiple classes of molecules such as lipids, metabolites, peptides, drugs, and their metabolites; a wide span of spatial resolution — from SIMS's nanometer scale to DESI's hundred-micrometer scale, adaptable to problems at different scales; and direct compatibility with spatial omics, integrating with transcriptomics and proteomics data.
Its limitations are equally clear: absolute quantification is affected by matrix effects and ionization efficiency differences; currently it is mostly semi-quantitative or relative distribution, and true quantification still requires internal standards and correction methods; matrix-requiring routes such as MALDI have matrix peak interference in the low-mass region (m/z<500), affecting the detection of small-molecule drugs and metabolites; vacuum routes impose higher requirements on sample preparation and the system. These are constraints that must be faced squarely during source selection and experimental design.
Spatial metabolomics is one of the fastest-growing directions in MSI, used to map the in-situ distribution of metabolites in tissues; drug distribution and metabolism research tracks the spatial distribution of candidate drugs and their metabolites within organs and tumors, serving as a powerful tool for tissue-level pharmacokinetic analysis; tumor boundary and heterogeneity analysis uses molecular maps to assist in identifying differences between paracancerous and cancerous tissue, providing clues for precise resection and classification; pathology and diagnosis serve as a molecular-level supplement to traditional pathological morphology.
Beyond research, MSI is gradually expanding into clinical pathology, forensic evidence, micro-localization of traditional Chinese medicine, environmental microplastic distribution, microbial colony metabolism, and other directions. With domestic imaging sources (such as Neo-Source LDPI/DPI) making breakthroughs in matrix-free, ambient, and single-cell resolution, the barrier to MSI adoption is falling.
To obtain detailed specifications, compatible models, or a quotation for the MSI LDPI / DPI full series imaging ion sources, visit the Neo-Source official website, or contact the official team for compatibility advice tailored to your mass spectrometer (Agilent / SCIEX / Thermo and other mainstream MS).