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Multi-Tiered µDicer Enables Protein-Preserving Microdissection at 10 µm Resolution
Annatoma Arif1, Rashmi Kumar2, Yumi Kwon2
1Department of Mechanical Engineering, Stanford University, Stanford, California, USA.
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.
Insights
Multi-tiered µDicers mechanically dissect tissues into microtissues for proteomic analysis. This method preserves more proteins than laser capture microdissection (LCM), especially at high spatial resolutions.
Area of Science:
- Biotechnology
- Proteomics
- Tissue Engineering
Background:
- Laser capture microdissection (LCM) is crucial for isolating regions of interest (ROIs) in tissues for molecular profiling.
- Challenges arise when ROIs approach cellular dimensions (~10 µm), as LCM can cause photothermal damage, compromising protein preservation and quantitative accuracy.
Purpose of the Study:
- To introduce multi-tiered µDicers, fabricated by two-photon polymerization, as a novel mechanical method for dissecting uniform microtissues down to 10 µm.
- To compare the proteomic yield and quality of microtissues generated by µDicers versus conventional LCM.
Main Methods:
- Fabrication of multi-tiered µDicers using two-photon polymerization.
- Mechanical dissection of ethanol-fixed human squamous cell carcinoma microtissues using µDicers and LCM.
- Proteomic analysis of microtissues using Nanodroplet Processing in One pot for Trace Samples (nanoPOTS) and liquid chromatography-mass spectrometry (LC-MS).
Main Results:
- µDicers yielded significantly more peptides and proteins than LCM, particularly at 10-20 µm spatial resolution.
- Confocal imaging revealed material loss (catapult-associated cavities) in LCM-generated microtissues, unlike µDicers.
- Multi-tiered µDicers enabled reproducible microdissection down to 10 µm while maintaining high protein coverage.
Conclusions:
- Multi-tiered µDicers offer a superior mechanical microdissection method compared to LCM for high-resolution spatial proteomic studies.
- This technology addresses limitations of LCM, improving protein preservation and quantitative fidelity in microtissues.
- µDicers have potential to complement LCM in next-generation spatial proteomic workflows.
