Feasibility Report on the Installation of an Imaging Ion Source on an Existing Agilent Mass Spectrometer to Achieve Mass Spectrometry Imaging Capabilities‌

This report evaluates the feasibility of retrofitting Agilent mass spectrometers (represented by the 6500 Series Q-TOF) 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. Evaluation conclusion: The Agilent platform possesses inherent advantages in terms of full-spectrum TOF acquisition, the standardisation of interfaces and operator familiarity; both the feasibility of retrofitting and the day-to-day user experience are deemed good.

I. Analysis of Agilent Platform Characteristics

The Agilent 6500 Series Q-TOF is currently one of the most widely used types of high-resolution mass spectrometers in Chinese universities and research institutions. Understanding its platform characteristics helps determine whether it can deliver the expected performance following the retrofitting of imaging capabilities.

Mass analyser characteristics. A quadrupole-time-of-flight (Q-TOF) hybrid analyser, the TOF section provides high resolution (typically 20,000–40,000 FWHM) and high mass accuracy (< 3 ppm as standard). For imaging applications, the TOF’s capability for simultaneous acquisition of the full spectrum is a key advantage—there is no need to preselect target ions; a single scan yields a spectrum covering the entire mass range, allowing for the flexible extraction of molecular images from any mass window during subsequent data processing.

Ion source interface. Agilent’s ion sources feature a push-fit flange design, with high machining precision for the positioning pins and sealing surfaces. The removal and installation of the ion source are standard procedures for routine maintenance, and operators are already familiar with the flange coupling process. This reduces the learning curve and the risk of operator error associated with installing the imaging source.

Software ecosystem. Agilent MassHunter is the standard data acquisition and processing software supplied with the instrument. The acquisition module directly supports full-scan mode; in imaging mode, a full spectrum is acquired for each pixel, and data files are in a universal format. For data processing, users can utilise MassHunter’s built-in functions or third-party imaging software (such as MSiReader), with clear data export paths.

Market penetration. A broad user base offers two practical benefits: firstly, there are numerous published case studies of imaging applications to draw upon; secondly, the technical support community is active, making it easier to find solutions when issues arise.

II. Item-by-item assessment of technical feasibility

2.1 Interface flange compatibility

The ion source interface flanges on the Agilent 6500 series are standardised in design. New Source provides dedicated adapter flanges for the Agilent platform; the physical dimensions, positioning method and O-ring specifications of these flanges correspond exactly to the host instrument’s interfaces.

Key points for adapter verification:

• Confirm the specific model of the host instrument (e.g. 6530, 6545, 6546, 6550, etc.) and year of manufacture

• Confirm the type of ion source currently in use (Jet Stream ESI, standard ESI, etc.)

• Provide photographs of the flange for model matching

The risk of incompatibility is low. Agilent ion source interfaces have maintained a high degree of backwards compatibility through many years of iteration, and flange specifications within the same generation remain largely unchanged.

2.2 Vacuum System

The additional load on the vacuum system following the installation of an imaging ion source is comparable to that of installing a standard ESI source. Agilent platforms are typically equipped with a two-stage vacuum system comprising a mechanical pump and a molecular pump; the fore-vacuum is maintained by the mechanical pump, whilst the high-vacuum stage is maintained by the molecular pump.

Attention should be paid to the frequency of vacuum switching. If frequent switching between the imaging source and the original ESI source is planned (for example, more than twice a week), the start-stop lifespan of the molecular pump must be assessed. Under normal operating conditions, the molecular pumps on Agilent platforms are typically designed to last 3–5 years; frequent venting and pumping cycles will accelerate bearing wear. The recommended strategy is to set fixed “imaging periods” (for example, using the imaging source for two consecutive days each week) to reduce the number of switchovers.

2.3 Mass Analyser

The analyser characteristics of the Q-TOF are ideally suited to imaging applications:

Full-spectrum acquisition. There is no need to decide which m/z values to monitor prior to imaging. A single scan covers the entire mass range, and once the data has been brought back, it can be analysed repeatedly to extract different molecular images from different mass channels. This feature is highly valuable in exploratory research—you may not know the distribution of a molecule corresponding to a particular m/z within a tissue, but as it is recorded in the data, it can be extracted for examination.

Scan speed. Agilent Q-TOF scan rates typically range from 10 to 50 Hz (depending on the mass range and resolution settings). In imaging scans with a spatial resolution of 100 μm, each pixel is held for 100–200 ms, allowing 2–10 full spectra to be acquired and averaged together, thereby ensuring a good signal-to-noise ratio.

Mass accuracy. The large number of pixels scanned in imaging mode places demands on the stability of mass calibration. It is recommended to perform calibration using standard samples prior to imaging experiments and to insert a calibration point at regular intervals during the experiment (for example, every 2 hours or every 2,000 pixels) for subsequent data correction.

2.4 Ion transfer efficiency

Ions generated by the imaging source enter the vacuum chamber from the atmospheric pressure side via the ion transfer capillary. The ion transfer design of the Agilent platform has been optimised over many years and offers high transfer efficiency. However, please note:

• As the position and angle of the spray needle in the imaging source differ from those of a standard ESI source, the spatial distribution of the ion plume will also vary. Following installation, it is recommended to adjust the capillary voltage and fragmentor voltage appropriately within the method to optimise the transmission efficiency for specific types of molecules.

• In DESI mode, the charge efficiency of the droplets generated by the spray differs from that of ESI; it may be necessary to set the capillary voltage 500–1000 V higher than for standard ESI to obtain an optimal signal.

2.5 Software Data Flow

Raw data files (.d format) generated by the MassHunter acquisition software in TOF-MS full-scan mode can be exported to a universal format using MassHunter Qualitative Analysis or third-party tools. The recommended data flow for imaging reconstruction is:

MassHunter acquisition → .d raw file

Convert to mzML or imzML format (using tools such as ProteoWizard msConvert)

Import into imaging software (MSiReader, Cardinal, SCiLS Lab, etc.) for reconstruction

This workflow has been validated in several published Agilent imaging studies and is proven and reliable.

III. Compatibility Assessment of Two Imaging Source Solutions

Solution 1: MSI DPI + Agilent Q-TOF

Option 2: MSI LDPI + Agilent Q-TOF

IV. Implementation Requirements

The installation of an imaging ion source on the Agilent platform requires the following conditions to be met at the implementation level:

Hardware requirements:

• The Q-TOF main unit is operating normally, with mass accuracy and sensitivity within the specified ranges

• The vacuum system is in good condition, with no significant decline in pumping speed

• There is space available for an injection pump (DPI) or laser optical path assembly (LDPI)

• A nitrogen supply is available (for DESI spray atomisation gas)

Personnel requirements:

• Operators must be able to independently carry out the removal and installation of the ion source

• Familiar with the basic operation of the MassHunter acquisition software

• Understanding of the basic principles of mass spectrometry imaging (can be learnt during installation training)

Time requirements:

• Installation: 1–2 days (flange installation + vacuum pumping + integrated commissioning)

• Training: 2–3 days (spray adjustment + sample scanning + hands-on practice of the entire data processing workflow)

• Trial operation: 1 week (completion of 2–3 full imaging experiments using the customer’s own samples)

V. Conclusions

The addition of an imaging ion source to the Agilent Q-TOF platform to enable mass spectrometry imaging is technically mature and feasible. The platform’s TOF full-spectrum acquisition capability, standardised flange interface and mature software ecosystem are its three core advantages. The primary concern is the long-term reliability of the vacuum system under conditions of frequent source switching; it is recommended to minimise the number of switches by establishing fixed imaging time slots.

Recommended approach: For routine tissue imaging, MSI DPI is the preferred choice; for applications requiring spatial resolution at the cellular level, MSI LDPI should be selected.