What are the steps of the MSI workflow?

Collection and preservation—sectioning and pretreatment—scanning acquisition—image reconstruction—validation registration—statistical interpretation, six stages in total, where any deviation propagates to the final image.

Where is the matrix-free source workflow simpler?

DESI/LDPI/DPI need no matrix spraying; you load the sectioned sample and go, compressing pretreatment from 'half a day to start' down to 'measure on the spot.'

How to choose section thickness?

Balance resolution and signal: too thick loses spatial resolution, too thin weakens signal; commonly 5–20 μm, to be optimized per source and analyte.

How are images compared with pathology?

Register the MSI section with adjacent H&E / immunohistochemistry sections, overlay the molecular signal onto the histological structure, and judge which cell type or region the signal falls in.

Search Query · Mass Spectrometry Imaging

What is the workflow of mass spectrometry imaging (MSI)?

A standard MSI workflow can be broken into six stages: 'collection—sectioning—(pretreatment)—scanning acquisition—image reconstruction—validation registration.' Any operational deviation at a stage propagates to the final image quality, so understanding the whole workflow matters more than knowing only the instrument.

Table of Contents
1. Collection and preservation 2. Sectioning and pretreatment 3. Scanning acquisition 4. Image reconstruction and validation registration

What is the workflow of mass spectrometry imaging (MSI)? — schematic diagram

1. Collection and preservation

Samples (tissue, plant, material) should be fixed or frozen as soon as possible after collection to prevent molecular degradation and distribution drift. Animal dosing studies need defined sampling time points so that 'drug distribution' can be mapped onto the pharmacokinetic time course.

Storage conditions (temperature, light protection, freeze-thaw avoidance) directly affect metabolite integrity. For clinical samples, ethics and traceability must also be addressed to ensure each section corresponds one-to-one with its pathology information.

2. Sectioning and pretreatment

Most MSI requires cutting the sample into serial thin sections (e.g., 5–20 μm), part for MSI and adjacent sections for H&E or immunohistochemistry validation. MALDI requires additional matrix spraying; matrix-free sources such as DESI/LDPI/DPI skip this step.

Section thickness affects spatial resolution and signal intensity (too thick loses resolution, too thin weakens signal). Neo-Source LDPI/DPI's matrix-free ambient route compresses pretreatment to 'section and load,' significantly reducing variables.

3. Scanning acquisition

The ion source scans the sample surface point by point at a set step size, acquiring a mass spectrum at every pixel to form a 'space × m/z × intensity' data cube. The step size determines resolution: LDPI can reach 2–3 μm, while DESI/DPI are in the tens to hundreds of micrometers.

Acquisition parameters (laser energy, spray solvent, photoionization power, mass range) need to be optimized for the analyte. Scanning should be performed under stable temperature/humidity and clean conditions to avoid air background and drift.

4. Image reconstruction and validation registration

After acquisition, software (SCiLS Lab, Cardinal, METASPACE, etc.) is used to reconstruct ion images, perform peak alignment, normalization and segmentation, and to register the molecular images with H&E / immunohistochemistry sections, overlaying signals onto the histological structure.

Finally, statistical differences are used to find molecular boundaries, combined with pathway annotation to give biological interpretation. The quality of the whole workflow depends on the reproducibility of the earlier steps, so it is advisable to establish SOPs and standard-material monitoring for each stage.

Frequently Asked Questions (FAQ)

What are the steps of the MSI workflow?: Collection and preservation—sectioning and pretreatment—scanning acquisition—image reconstruction—validation registration—statistical interpretation, six stages in total, where any deviation propagates to the final image.
Where is the matrix-free source workflow simpler?: DESI/LDPI/DPI need no matrix spraying; you load the sectioned sample and go, compressing pretreatment from 'half a day to start' down to 'measure on the spot.'
How to choose section thickness?: Balance resolution and signal: too thick loses spatial resolution, too thin weakens signal; commonly 5–20 μm, to be optimized per source and analyte.
How are images compared with pathology?: Register the MSI section with adjacent H&E / immunohistochemistry sections, overlay the molecular signal onto the histological structure, and judge which cell type or region the signal falls in.

Get Specifications & Quotation

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).

Tissue Sectioning (Mass Spectrometry Imaging Sample Preparation)Frozen Sectioning (Mass Spectrometry Imaging)Matrix Spraying and Matrix-free Single-Cell Mass Spectrometry Imaging imzML Mass Spectrometry Imaging Data Standard Format Pathology Imaging: Overlaying a Molecular Map Under the Microscope

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