Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion-mobility spectrometry (TIMS) imaging platform. This prototype MALDI timsTOF instrument is capable of 10 μm spatial resolutions and 20 pixels/s throughout molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 ×10 μm at the sample surface. General image performance was assessed using murine kidney and brain *

Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct mapping of biomolecules in tissue. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables approximately a ~270% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn-backbone isomers, acyl chain isomers, as well as double bond positional and geometric isomers are demonstrated. As a proof-of-concept, in situ separation and imaging of lipid isomers with distinct spatial distributions was performed using tissue sections from a whole-body mouse pup. File list (2) download file view on ChemRxiv 200813_Manuscript_v2_FINAL.pdf (756.02 KiB) download file view on ChemRxiv 200813_Supplemental_v2_FINAL.pdf (1.08 MiB)

Imaging mass spectrometry has become a mature molecular mapping technology that is used for molecular discovery in many medical and biological systems. While powerful by itself, imaging mass spectrometry can be complemented by the addition of other orthogonal, chemically informative imaging technologies to maximize the information gained from a single experiment and enable deeper understanding of biological processes. Within this review, we describe MALDI, SIMS, and DESI imaging mass spectrometric technologies and how these have been integrated with other analytical modalities such as microscopy, transcriptomics, spectroscopy, and electrochemistry in a field termed multimodal imaging. We explore the future of this field and discuss forthcoming developments that will bring new insights to help unravel the molecular complexities of biological systems, from single cells to functional tissue structures and organs.