Isomer-Selective Mass Spectrometry Imaging Using Nanospray Desorption Electrospray Ionization (Nano-DESI)

Mass spectrometry imaging (MSI) has transformed our ability to explore molecular distributions in biological tissues with high chemical specificity and sensitivity. Despite significant advances in this field, the absence of separation prior to analysis leads to isomeric and isobaric overlaps, posing a major analytical challenge. To enhance chemical specificity and enable isomer differentiation, tandem mass spectrometry, ion mobility spectrometry, chemical complexation, and derivatization strategies are increasingly integrated into MSI workflows. Ambient ionization MSI techniques provide both chemical and spatial information under native or near-native conditions, enabling rapid, label-free molecular imaging of complex biological samples with minimal sample pretreatment. Among the most promising ambient MSI techniques is nanospray desorption electrospray ionization (nano-DESI), a method that relies on localized liquid extraction directly from biological tissue sections. We have successfully implemented custom-designed nano-DESI platforms on multiple commercial mass spectrometers to enable molecular identification at each pixel of the image and facilitate isomer-selective mass spectrometry imaging (iMSI).This Account highlights recent advances in iMSI using nano-DESI. Key developments include the integration of nano-DESI with multiple reaction monitoring on a triple quadrupole mass spectrometer to differentiate isomeric lipids in biological tissues. We also describe the integration of photoinitiated derivatization and metal ion complexation strategies to enable isomer-selective imaging using structure-specific fragments generated by collision induced dissociation. Furthermore, high-resolution separation of lipid isomers was achieved by coupling nano-DESI with trapped ion mobility spectrometry, demonstrating the value of gas-phase separation for iMSI. These innovations have significantly expanded the analytical capabilities of MSI critical to probing the spatial organization of isomeric lipids and metabolites in biological systems. We also discuss future directions, including new complexation strategies and the integration of nano-DESI with data-independent acquisition and parallel accumulation serial fragmentation technologies. Collectively, these advances establish nano-DESI iMSI as a powerful and versatile tool in the evolving field of spatial metabolomics and lipidomics.