Un μDicer multinivel con cuchillas jerárquicas logra la microdisección que preserva proteínas hasta 10 μm

To study tissue heterogeneity at the sub-millimeter scale, laser capture microdissection (LCM) has been the leading technology for isolating regions of interest (ROI) for downstream molecular profiling. As the ROI approaches cellular dimensions (∼10 μm), laser-induced photothermal damage and challenges in capturing microtissues in conventional LCM can compromise protein preservation and quantitative fidelity. This work introduces multi-tiered μDicers, fabricated by two-photon polymerization, to mechanically dissect tissue slices into uniform microtissues down to 10 μm. The hierarchical blade architecture limits instantaneous blade-tissue engagement and lowers the cutting force relative to single-tier designs. For benchmarking, proteomic analysis is performed on ethanol-fixed human squamous cell carcinoma microtissues generated by μDicers and by LCM. Under identical Nanodroplet Processing in One pot for Trace Samples (nanoPOTS) and liquid chromatography-mass spectrometry (LC-MS) conditions, μDicers yield more peptides and proteins than LCM, with the largest gains at 10-20 μm spatial resolution. Confocal imaging shows catapult-associated cavities in LCM-generated microtissues. This material loss, along with membrane-limited protein extraction, likely reduces protein coverage. In contrast, multi-tiered μDicers enables reproducible microdissection down to 10 μm while maintaining high protein coverage. With spatial registration of microtissues under development, μDicers have potential to complement LCM for next-generation spatial proteomic workflows.