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.

All MALDI IMS data were acquired using a timsTOF fleX (Bruker Daltonics, Bremen, Germany).⁸⁸ 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 m/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= m/z 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.