Enhancing the Sensitivity of Mass Spectrometry Imaging through Spatial Signal Averaging

Mass spectrometry imaging (MSI) enables spatially resolved molecular analysis, but automatic data reduction imposed by instrument software, such as in Orbitrap systems, can severely limit the detection of low-abundance ions. When these ions are targeted, the resulting sparse detection hinders spatial interpretation and compromises quantitative accuracy. Using an external high-performance data acquisition system to capture full-length transients, we demonstrate that adjacent-pixel averaging of full-profile (unreduced) Orbitrap data markedly improves the detection frequency of low-abundance peaks without affecting the detection of highly abundant species in biological tissue samples. Pixel averaging increased the coverage of low-abundance ions from less than 10% to over 90% in mouse brain and nonhuman primate vaginal tract tissues. Quantitative accuracy was also enhanced, as shown by improved agreement between observed and theoretical isotopic ratios, and between MSI-derived and liquid chromatography (LC)–MS/MS measured total analyte concentrations in adjacent tissue sections. These findings establish pixel averaging as a simple yet powerful strategy to extend MSI sensitivity and improve quantitative biochemical mapping in complex tissues, while remaining fully compatible with other sensitivity-enhancing techniques.