Multi‐Tiered µDicer Enables Protein‐Preserving Microdissection at 10 µm Resolution

ABSTRACT Laser capture microdissection (LCM) remains the leading technology for isolating sub‐millimeter regions of interest (ROIs) to enable downstream molecular profiling and study tissue heterogeneity. 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 enable reproducible microdissection down to 10 µm while maintaining high protein coverage. With the spatial registration of microtissues under development, µDicers have the potential to complement LCM for next‐generation spatial proteomic workflows.