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.
Desyaterik et al. (2026) studied this question.