Feasibility Report on the Installation of an Imaging Ion Source on an Existing AB SCIEX Mass Spectrometer to Achieve Mass Spectrometric Imaging Capabilities‌

This report assesses the feasibility of retrofitting AB SCIEX mass spectrometers (represented by the TripleTOF series) with the Neo Source INSTRUMENT MSI DPI or MSI LDPI imaging ion sources, based on five dimensions: platform characteristics, interface compatibility, analyser capabilities, software ecosystem and operational model. Conclusion of the assessment: The high-speed acquisition capabilities and stable ion source interface design of the AB SCIEX platform provide a solid foundation for imaging applications; retrofitting is feasible.

I. Analysis of AB SCIEX Platform Characteristics

The AB SCIEX TripleTOF series comprises high-performance Q-TOF mass spectrometers, which are widely used in the fields of analytical chemistry, metabolomics and drug discovery in China due to their high scanning speed and high sensitivity.

Mass analyser characteristics. The name ‘TripleTOF’ derives from its triple TOF design—a configuration that delivers high mass resolution (typically 30,000–40,000 FWHM) and excellent mass accuracy (< 2 ppm under standard conditions). For imaging applications, this high-resolution, high-accuracy mass analysis capability ensures greater reliability in spatial molecular identification.

Advantages in scanning speed. A standout feature of the TripleTOF is its high acquisition rate (typically up to 100 Hz or higher, depending on the mass range). In imaging scans, the dwell time per pixel is typically in the order of 50–200 ms. The TripleTOF’s scanning speed allows for the accumulation of more spectra per pixel within the same time frame for averaging, or for covering a larger area with the same number of averaging passes.

Ion source interface. AB SCIEX employs a Turbo V ion source interface design, featuring a proprietary gas curtain structure. The gas curtain forms an inward-flowing barrier at the ion inlet, preventing neutral molecules and solvent clusters from entering the vacuum chamber whilst allowing ions to be guided in by the electric field. This design effectively reduces background noise during routine analysis and is equally effective in imaging applications.

Software ecosystem. Analyst TF is the standard acquisition and processing software for the TripleTOF. The acquisition module supports TOF-MS full-scan mode, whilst the data processing module provides Multiquant for quantitative analysis and PeakView for qualitative analysis. The reconstruction of imaging data is typically carried out using third-party software.

II. Item-by-item assessment of technical feasibility

2.1 Interface Flange Adaptation

The design of the AB SCIEX Turbo V ion source interface differs from Agilent’s push-fit flange, utilising a mounting method featuring positioning studs and a locking knob. New Source provides a dedicated adapter flange for the AB SCIEX platform, with flange dimensions and positioning structure fully compatible with the Turbo V interface.

Key points for adaptation verification:

• Confirm the specific model of the main unit (e.g. TripleTOF 5600, 6600, 7600, etc.)

• Confirm the type of ion source currently in use (Turbo V ESI, DuoSpray, etc.)

• Provide photographs of the flange interface

The risk of incompatibility is low. The Turbo V interface specifications are standardised across all sub-models of the AB SCIEX TripleTOF series, ensuring good physical compatibility.

2.2 Air Curtain Gas Adaptation

The air curtain gas design, unique to the AB SCIEX platform, requires special attention when installing an imaging source. The flow rate and pressure of the air curtain gas are automatically controlled by the main unit; however, following the installation of the imaging source, changes to the physical dimensions of the flange interface may slightly affect the flow field distribution of the air curtain gas.

Impact Assessment:

• The flow parameters of the air curtain remain unchanged (typically 25–35 psi) and are set by the main unit software

• The changes to the flow field introduced by the flange connection are minimal, typically falling within the normal fluctuation range

• When conducting spray tests after installation, observe whether there are any significant changes in the background noise level. If the background noise increases significantly (TIC baseline rises by more than 50 per cent), check the flange seal

• Expected impact: Virtually negligible. The Turbo V’s air curtain design offers good flow field robustness and is not sensitive to minor geometric variations in the interface flange

It is recommended that the following item be added to the acceptance testing: compare the background noise and signal-to-noise ratio before and after the installation of the imaging source by performing a routine direct injection (via the ESI source) using a standard sample to confirm that there are no significant changes.

2.3 Mass Analyser

The TripleTOF analyser performs exceptionally well in imaging applications:

High scanning speed. A scanning speed of 100 Hz means that approximately 10 full spectra can be acquired within a pixel dwell time of 100 ms. Averaging these 10 spectra can improve the signal-to-noise ratio by approximately 3.2 times (theoretical value: √10), effectively enhancing the imaging quality of low-abundance molecules.

High resolution. A resolution of 35,000–40,000 is sufficient to distinguish between most isotopic interferences. In complex biological tissue imaging samples (rich in lipids and metabolites), this level of resolution can effectively reduce false positives caused by peak overlap.

Mass accuracy stability. The TripleTOF’s built-in automatic calibration system (CDS, Calibrant Delivery System) is particularly useful in imaging experiments. Imaging scans may last for several hours, during which changes in ambient temperature may cause mass axis drift. When the CDS function is enabled, the instrument periodically introduces a calibration solution to correct the mass axis, ensuring stable mass accuracy during prolonged imaging scans.

2.4 Data Flow

Raw data files (.wiff and .wiff.scan) generated by the Analyst TF acquisition software can be converted to mzML format using the MS Data Converter tool provided by AB SCIEX or third-party tools (such as ProteoWizard). Recommended data processing workflow:

1. Analyst TF acquisition → .wiff files

2. Conversion using MS Data Converter → mzML

3. mzML encapsulation (addition of spatial coordinate information)

4. Import into imaging software to reconstruct molecular images

It is recommended to confirm prior to purchase whether the MS Data Converter tool is already installed and whether additional licences are required (some versions are available free of charge).

2.5 Ion Transfer Efficiency

The gas curtain design of the Turbo V interface performs exceptionally well in blocking neutral noise; however, this also means that ions must pass through this gas curtain before entering the vacuum chamber. The gas curtain is more effective at blocking ions with low mass numbers than those with high mass numbers. In imaging scenarios, if the target analytes are small-molecule metabolites (m/z < 200), the signal may be weaker than that for large molecules (m/z > 400). This is a characteristic of the platform rather than an issue with the imaging source, and should be taken into account during data analysis.

III. Compatibility Assessment of Two Imaging Source Solutions

Solution 1: MSI DPI + AB SCIEX TripleTOF

Option 2: MSI LDPI + AB SCIEX TripleTOF

IV. Implementation Requirements

The requirements for installing an imaging ion source on the AB SCIEX platform are essentially the same as those for the Agilent platform. Additional points to note:

Stock of CDS calibration solutions. If you plan to utilise the CDS function to maintain mass accuracy during long-duration imaging scans, ensure that there is a sufficient supply of calibration solutions (APCI positive and negative ion calibration solutions). A set of calibration solutions typically lasts for several days.

Data storage. The volume of TripleTOF WFIFT data files increases rapidly at high scan speeds. For a single imaging scan covering a 5 mm × 5 mm area at 50 μm resolution (approximately 10,000 pixels), if 10 full spectra are acquired per pixel, the raw data volume may be in the order of 5–10 GB. Data storage and backup arrangements must be confirmed in advance.

V. Conclusions

Fitting an imaging ion source to the AB SCIEX TripleTOF platform is technically feasible. The platform’s two key advantages—the improved pixel signal-to-noise ratio resulting from high scanning speeds, and the long-term stability of scan quality provided by CDS automatic calibration—enable it to perform exceptionally well in imaging experiments requiring large areas and high pixel density.

Recommended approach: Select MSI DPI for routine tissue imaging scenarios, and MSI LDPI for high-pixel-density, high-resolution imaging scenarios (the speed advantage of TripleTOF is more valuable in this context).