DOI: 10.5061/dryad.bnzs7h4jz
title: Data from: In situ lipidomics of Staphylococcus aureus osteomyelitis using imaging mass spectrometry

description: Osteomyelitis occurs when Staphylococcus aureus invades the bone
 microenvironment, resulting in a bone marrow abscess with a spatially
 defined architecture of cells and biomolecules. Imaging mass spectrometry
 and microscopy are invaluable tools that can be employed to interrogate
 the lipidome of S. aureus-infected murine femurs to reveal metabolic and
 signaling consequences of infection. Here, nearly 250 lipids were
 spatially mapped to healthy and infection-associated morphological
 features throughout the femur, establishing composition profiles for
 tissue types. Ether lipids and arachidonoyl lipids were significantly
 altered between cells and tissue structures in abscesses, suggesting their
 roles in abscess formation and inflammatory signaling. Sterols,
 triglycerides, bis(monoacylglycero)phosphates, and gangliosides possessed
 ring-like distributions throughout the abscess, suggesting a hypothesized
 dysregulation of lipid metabolism in a population of cells that cannot be
 discerned with traditional microscopy. These data provide chemical insight
 into the signaling function and metabolism of cells in the fibrotic border
 of abscesses, likely characteristic of lipid-laden macrophages.
description: All MALDI IMS data were acquired using a timsTOF fleX (Bruker
 Daltonics, Bremen, Germany).<sup>88</sup> All imaging
 experiments were operated in qTOF mode with TIMS deactivated unless
 otherwise noted. For acquisition of the main dataset, a pitch offset
 strategy was employed. The SmartBeam 3D 10 kHz frequency tripled Nd:YAG
 laser (355 nm) was focused and beam scan activated to produce a 10 μm x 10
 μm burn pattern. The z position of the stage was adjusted to keep the
 laser focused and account for additional height of the mount and Cryofilm.
 Positive ion data were acquired first with a stage pitch of 20 μm. Stage
 coordinates in flexImaging (Bruker Daltonics) were then offset 10 μm in
 each x-y dimension, and negative ion data were acquired with the same 20
 μm stage pitch. This strategy yields an effective spatial resolution of 20
 μm for both polarities from the same pixel regions. For high spatial
 resolution (10 μm) experiments, beam scan was still implemented, but the
 stage pitch was set to 10 μm. The laser was set to 80% power (0%
 attenuator offset) and 100 shots. Prior to data acquisition in positive
 and negative ion modes, ESI-L Tune Mix (Agilent Technologies, Santa Clara,
 CA, USA) was infused into the system for external mass calibration. For
 both polarities, data were acquired from <em>m/</em>z
 150-2000. For positive ion mode, the following MS1 parameters were set:
 Transfer- MALDI Plate Offset= 30.0 V, Deflection 1
 Delta= 70.0 V, Funnel 1 RF= 450.0 Vpp, isCID Energy= 0.0 eV, Funnel 2 RF=
 500.0 Vpp, Multipole RF= 500.0 Vpp; Collision Cell-
 Collision Energy= 10.0 eV, Collison RF= 2900.0 Vpp;
 Quadrupole- Ion Energy= 5.0 eV, Low Mass=
 <em>m/z</em> 300.00; Focus Pre TOF-
 Transfer Time= 110.0 µs, Pre Pulse Storage= 10.0 µs. For negative ion
 mode, changes include: MALDI Plate Offset= -30.0 V, Deflection 1 Delta=
 -70.0 V, Collision Energy= -10.0 eV, Ion Energy= -5.0 eV.
description: # Data from: In situ lipidomics of Staphylococcus aureus osteomyelitis
 using imaging mass spectrometry
 [https://doi.org/10.5061/dryad.bnzs7h4jz](https://doi.org/10.5061/dryad.bnzs7h4jz) Included are all imaging mass spectrometry datasets used for the main figures of this manuscript Also included is a spreadsheet entitled "PCCValues(Figure7)" which gives the raw PCC values from Figure 7. ## Description of the data and file structure All data are in vendor-neutral imzml format, which is common for imaging mass spectrometry datasets. A cohort of three infected mice and three mock-infected mice were used for the main dataset involved in the majority of experimental analyses (**Figures 1-6**). Additional Catchup mice were used for the high spatial resolution imaging experiments (**Figure 7**). For the main dataset, the 9 infected sections (3 section replicates for 3 biological replicates-mice) are located in "imzmls-Infected" folder. The 9 mock-infected sections are located in "imzmls-mock" folder. Each folder has positive and negative data clearly labeled. The section labels are as followed: culumlative#dataset)#mouse-#sectionreplicate"m or i""pos or neg". Example: 1)1-1mneg is first overall dataset, first mouse, first section replicate of that mouse, mock, neg ion mode. For the catchup mouse experiment, the four datasets are located in "catchupimzmls," and are organized by infection status and pos/neg polarity. All folders are zipped to minimize size. All data were exported from SCiLS Lab v2023 (Bruker Daltonics) with root mean square normalization preserved. ## Accessing data .imzml files and their associated .ibd files can be opened with commercial imaging software, SCiLS (Bruker Daltonics). If one does not have a license to use this software, they can run vendor-neutral scripts and software to visualize and mine the data. A wide variety of options are summarized here: [https://www.ms-imaging.org/imzml/software-tools/](https://www.ms-imaging.org/imzml/software-tools/). The authors would suggest to start with Cardinal ([https://cardinalmsi.org/](https://cardinalmsi.org/) ) due to their previous experience with this R package.