ABSTRACT: While decades of technical and analytical advancements have been utilized to discover novel lipid species, increase speciation, and evaluate localized lipid dysregulation at sub-tissue, cellular and sub-cellular levels, many challenges still exist. One limitations is that the acquisition of both in-depth spatial information and comprehensive lipid speciation is extremely difficult, especially when biological material is limited or lipids are at low abundance. In neuroscience, for example, it is often desired to focus on only one brain region or sub-region, which can be well under a square mm for rodents. Herein, we evaluate a micropunch histology method where cortical brain tissue at 2.0, 1.25, 1.0, 0.75, 0.5 and 0.25 mm diameter sizes and 1mm depth was collected and analyzed with multidimensional liquid chromatography, ion mobility spectrometry, collision induced dissociation and mass spectrometry (LC-IMS-CID-MS) measurements. Lipid extraction was optimized for the small sample sizes and assessment of lipidome coverage for the 2.0 to 0.25 mm diameter sizes showed a decline from 304 to 198 lipid identifications as validated by all 4 analysis dimensions (~35% loss in coverage for ~88% less tissue). While losses were observed, the ~200 lipids and estimated 4,630 neurons contained within the 0.25 punch still provided in-depth characterization of the small tissue region. Furthermore, while localization routinely achieved by mass spectrometry imag-ing (MSI) and single cell analyses is greater, this diameter is sufficiently small to isolate sub-cortical, hypothalamic, and other brain regions in adult rats, thereby increasing the coverage of lipids within relevant spatial windows without sacrificing speciation. Therefore, micropunch histology enables in-depth, region-specific lipid evaluations, an approach that will prove beneficial to a variety of lipidomic applications.
Erin Baker 
