Mar 23, 2026

High Spatial Resolution MALDI IMS Data Acquisition for Metabolites in Negative Ion Mode Using timsTOF Flex

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo12oxg4o/v1
  • Ali Zahraei1,
  • Madeline E. Colley1,
  • Martin Dufrense1,
  • Melissa Farrow1,
  • Jeff Spraggins1
  • 1Vanderbilt University
  • VU Biomolecular Multimodal Imaging Center / Spraggins Research Group
    Tech. support email: [email protected]
Ali Zahraei
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo12oxg4o/v1
Protocol Citation: Ali Zahraei, Madeline E. Colley, Martin Dufrense, Melissa Farrow, Jeff Spraggins 2026. High Spatial Resolution MALDI IMS Data Acquisition for Metabolites in Negative Ion Mode Using timsTOF Flex. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo12oxg4o/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 13, 2026
Last Modified: March 23, 2026
Protocol  Integer ID: 243288
Keywords: imaging mass spectrometry, metabolite imaging, high spatial resolution maldi ims data acquisition, mass spectrometry, negative ionization mode, µm spatial resolution, metabolite, negative ion mode, bruker timstof flex instrument, performing maldi, instrument parameter, µm
Abstract
This protocol describes the workflow and instrument parameters for performing MALDI imaging mass spectrometry (IMS) using a Bruker timsTOF Flex instrument. The method is optimized for metabolite imaging at 10 µm spatial resolution, covering an m/z range of 50–900, acquired in negative ionization mode using qTOF operation.
Materials
Red phosphorus
ESI-L LCMS Tuning Solution (Agilent)
Flatbed scanner
Load the MTP slide adapter 2 into the instrument. Open timsControl software and select the appropriate MALDI IMS data acquisition method. Measure the target height at multiple locations across the slide and verify that height variation is less than 10 µm.

  • Note: Representative acquisition parameters for 10 µm spatial resolution MALDI IMS include: m/z range 50 - 900, laser power 50–60%, laser frequency 10,000 Hz, 100 - 200 laser shots per pixel, 10 µm spot size with beam scanning disabled, and a source temperature at 50 °C.
Perform external TOF calibration using red phosphorus. Confirm that all calibration points meet acceptance criteria of <1 ppm mass error and a calibration score >99%. Do not proceed with data acquisition if calibration criteria are not met.

  • Note: Red phosphorus is typically used for MALDI calibration. Alternatively, ESI-L LCMS Tuning Solution (Tuning Mix, Agilent) can be used for calibration when operating the instrument in electrospray ionization (ESI) mode.

List of Red Phosphorus Mass Calibration Peaks in negative mode
A
Negative mode
278.7644
402.6594
464.6069
526.5545
650.4495
774.3445
898.2396
Use FlexImaging software to load the optical image of the slides. Define the target position using three teaching points. Verify teaching accuracy by moving the stage to a fourth reference location and confirming accurate alignment between the stage position and optical image.
Define the tissue measurement region by creating a region of interest (ROI) in FlexImaging software. Confirm accurate ROI placement relative to tissue boundaries and pixel or raster size. Start data acquisition within FlexImaging.