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

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.