Development of a Tapping-Mode Scanning Probe Electrospray Ionization Platform for High-Sensitivity and Long-Term Stability in Single-Cell Mass Spectrometry Imaging of Tissue.

To elucidate disease mechanisms, it is essential to develop analytical techniques capable of visualizing cellular heterogeneity in biological tissues. Mass spectrometry imaging (MSI) allows simultaneous visualization of multiple biomolecules in tissue sections in a single measurement. However, achieving single-cell (SC)-MSI requires high ion detection sensitivity and long-term ionization stability. Tapping-mode scanning probe electrospray ionization (t-SPESI) delivers a tiny amount of solvent from an oscillating fused silica capillary probe to the sample surface, enabling rapid extraction and ionization of analytes. In this study, we developed a t-SPESI measurement system and a chemical modification method for the probe surface to enable SC-MSI of biological tissues. Introducing a compact sample stage shortened the ion transfer tube to the mass spectrometer, thereby increasing ion detection sensitivity. Furthermore, forming a fluorine-containing molecular layer on the probe surface suppressed biomolecule adsorption at the probe tip and increased the long-term stability of solvent delivery. SC-MSI of mouse brain sections was performed at pixel sizes of 10 and 5 μm and visualized lipid distributions corresponding to fine tissue structures. These results demonstrate that the combination of system miniaturization and probe surface modification is an effective strategy for achieving high-sensitivity and long-term stable SC-MSI via t-SPESI, providing a robust platform for investigating cellular heterogeneity in biological tissues.