Feasibility Report on the Implementation of Mass Spectrometry Imaging Using an Imaging Ion Source Fitted to an Existing Thermo Mass Spectrometer‌

This report assesses the feasibility of retrofitting Thermo brand mass spectrometers (represented by the Q Exactive series Orbitrap platform) with Neo Source INSTRUMENT’s MSI DPI or MSI LDPI imaging ion sources, based on five dimensions: platform characteristics, interface compatibility, analyser capabilities, software ecosystem and operational model. Evaluation conclusion: The ultra-high resolution and exceptional mass accuracy of the Thermo Orbitrap platform provide robust support for molecular identification in imaging data; retrofitting is feasible. However, attention must be paid to the balance between scan speed and imaging throughput in ultra-high resolution mode.

I. Analysis of Thermo Platform Characteristics

The Thermo Q Exactive series (including the Q Exactive, Q Exactive Plus, Q Exactive HF, Q Exactive HF-X, etc.) is one of the most widely used Orbitrap platforms in the field of high-resolution mass spectrometry, with a large user base in proteomics, metabolomics, drug metabolism and food safety.

Mass analyser characteristics. The core advantage of the Orbitrap lies in its resolution—the Q Exactive series offers ultra-high resolution ranging from 70,000 to 280,000 FWHM (depending on the model and mass number), with mass accuracy typically < 1 ppm. In imaging applications, this means:

• Strong capability for molecular identification of complex mixtures such as lipids and metabolites

• Effective separation even in the presence of closely adjacent isotope peaks of equal intensity

• A lower rate of ‘false positives’ in imaging data

Scanning speed characteristics. The resolution of an Orbitrap is inversely proportional to its scanning speed—the higher the resolution, the longer a single scan takes. This is a key trade-off to consider in imaging applications. At a resolution of 70,000, the Q Exactive has a scan rate of approximately 3–4 Hz; at a resolution of 140,000, it is approximately 1.5–2 Hz. By comparison, TOF-type instruments typically have scan rates in the range of 10–100 Hz.

This means that in imaging scenarios, if high-resolution identification accuracy is prioritised, the number of spectra per pixel will be lower than on TOF platforms. It is necessary to consider this carefully during the experimental design phase: is the core output of this experiment to “know precisely which molecule is present at each location” (a strength of the Orbitrap), or to “rapidly cover a large area to obtain the spatial distribution of molecules” (an area where TOF excels)?

Ion source interface. The Q Exactive series utilises HESI (heated electrospray ionisation) or standard ESI ion sources. The ion source interface features a flanged mounting design, secured via a mechanical locking mechanism. In imaging applications, the heating function of the HESI source can be used to assist in the desolvation of DESI sprays.

Software ecosystem. Thermo Xcalibur is the standard acquisition and processing software, whilst FreeStyle is used for qualitative data processing. Third-party software is required for imaging data reconstruction.

II. Item-by-item assessment of technical feasibility

2.1 Interface flange adapters

The ion source interface flanges of the Q Exactive series differ from those of Agilent and AB SCIEX in terms of physical dimensions and positioning methods. New Source provides dedicated adapter flanges for the Thermo platform.

Key points for adaptation verification:

• Confirm the full model of the main unit (Q Exactive, Q Exactive Plus, HF, HF-X, etc.)

• Confirm the current ion source type (HESI, standard ESI, Nanospray, etc.)

• Provide photographs of the flange interface

The risk of incompatibility is low. The specifications of the ion source interface flanges are essentially consistent across all Q Exactive sub-models.

2.2 Utilisation of the HESI heating function

The heating function of the HESI source is used to assist desolvation during standard ESI operations. After fitting an imaging source, the HESI heating function (auxiliary gas heating) is no longer used directly for spraying (as spraying is performed by the DESI spray needle of the imaging source), but it can still be used to heat the ion transfer tube, aiding the desolvation of ions before they enter the vacuum.

It is recommended to enable ion transfer tube heating (standard temperature 250–320 °C) during imaging experiments, as this helps to improve the desolvation efficiency of the charged droplets generated by DESI spraying.

2.3 Ion Transfer Path

The ion transfer path in the Thermo Q Exactive is relatively complex: atmospheric pressure ions → Ion Transfer Tube → S-Lens (RF ion focusing) → Injection Flatapole → Bent Flatapole → Quadrupole mass filter → C-Trap → Orbitrap.

The multi-stage ion optical path has two main effects on ion transport from imaging sources:

• Advantage: The multi-stage ion optics provide a wealth of ion transport adjustment parameters (S-Lens RF Level, Injection Time, etc.), allowing for fine-tuning to optimise the characteristics of ions generated by imaging ion sources

• Note: Due to the longer ion transport path and greater number of stages, ion loss is slightly higher than in TOF platforms with shorter paths. This manifests as the absolute signal intensity of the Orbitrap potentially being lower than that of the TOF at the same analyser concentration. This must be taken into account in low-abundance imaging analyses

2.4 The trade-off between resolution and scan speed

This is the most critical engineering decision point in imaging applications on the Orbitrap platform.

A few points of analysis:

• At resolutions of 35,000–70,000, the imaging throughput is acceptable. This resolution level is already higher than that of most TOF platforms and is sufficient for reliable molecular identification

• Ultra-high resolutions of 140,000 and above are suitable for small-area (< 2 mm × 2 mm), high-value imaging experiments, such as the detailed molecular mapping of tumour boundaries

• If the primary objective is to obtain images of molecular spatial distribution rather than the precise identification of each molecule, a resolution of 17,500–35,000 is sufficient

2.5 Data Flow

The data flow for the Thermo platform is as follows:

1. Xcalibur acquisition → .raw files

2. Conversion to mzML using ProteoWizard msConvert or Thermo RawFileReader

3. Molecular image reconstruction using imaging software

Please note the compatibility of data extraction tools for .raw files. ProteoWizard may experience compatibility issues with certain versions of the Thermo format; it is recommended that you test the conversion process using existing .raw files from your laboratory prior to purchase. If ProteoWizard does not support the current version of the .raw format, you may use the MSFileReader library provided by Thermo or export the data using FreeStyle software.

III. Compatibility Assessment of Two Imaging Source Solutions

Solution 1: MSI DPI + Thermo Q Exactive

Option 2: MSI LDPI + Thermo Q Exactive

IV. Implementation Requirements

Special considerations regarding the installation of an imaging source on the Thermo platform:

Estimated experiment duration. Compared with other platforms, Orbitrap imaging experiments take longer for the same area. Taking a 5 mm × 5 mm area with a resolution of 50 μm as an example: approximately 10,000 pixels, with a dwell time of 200 ms per pixel, results in a total scan time of approximately 33 minutes. If a resolution of 140,000 is used (with a dwell time of 500 ms per pixel to accumulate sufficient scan repetitions), the total time is approximately 83 minutes. This factor must be taken into account when planning experiments.

Optimisation of S-Lens parameters. After installing the imaging source, it may be necessary to adjust the S-Lens RF level. In imaging mode, the spray position and ion plume morphology differ from those in standard ESI, and the optimal RF value for the S-Lens may vary. It is recommended to carry out a minor optimisation of the S-Lens RF level during the acceptance testing phase.

Data file size management. .raw files are larger than the .d or .wiff files on TOF platforms (typically 1.5–2 times larger). A typical imaging experiment may generate 10–20 GB of data.

V. Conclusions

The installation of an imaging ion source on the Thermo Q Exactive platform is technically feasible. The platform’s core strengths—ultra-high resolution and outstanding mass accuracy—make it invaluable for research requiring precise molecular identification of imaging data.

The trade-off between Orbitrap scanning speed and imaging throughput must be considered during the experimental design phase, and resolution parameters should be selected appropriately based on the experimental objectives (coverage or identification depth).

Recommended approach: For tissue imaging scenarios requiring precise molecular identification, select MSI DPI. The LDPI approach on the Orbitrap is suitable for fine-scale imaging of small areas; for large-area applications, the time cost must be assessed.