研究目的
To resolve the fine electromechanical structure of collagen fibrils at the nanoscale by overcoming challenges such as weak piezoresponse, rough topography, and hierarchical microstructure using sequential excitation piezoresponse force microscopy (SE-PFM).
研究成果
SE-PFM, combined with PCA and SHO model, successfully resolves the fine electromechanical structure of collagen fibrils, revealing heterogeneity correlated with gap and overlap domains. This approach provides high-fidelity mappings and quantitative analysis, overcoming limitations of conventional techniques like DART-PFM, and can be extended to other applications in big data AFM.
研究不足
The method requires multiple scans, leading to longer experimental times. It may not be fully reliable under very large topography variations, and the weak piezoresponse of biological systems poses challenges for quantitative measurement. The applicability to other biological materials with different microstructures is not fully explored.
1：Experimental Design and Method Selection:
The study uses sequential excitation (SE) piezoresponse force microscopy (PFM) to excite collagen samples with a sequence of distinct AC frequencies, minimizing crosstalk with topography. Data is processed using principal component analysis (PCA) to remove noise and simple harmonic oscillator (SHO) model for physical analysis.
2：Sample Selection and Data Sources:
Type I collagen samples are processed from fresh porcine artery, specifically from the adventitia layer. Samples are fixed in paraformaldehyde, embedded, frozen, and sliced into 10 μm thickness sections.
3：List of Experimental Equipment and Materials:
Equipment includes a Cypher-ES AFM (Asylum Research), ARROW-CONTPt cantilevers (spring constant
4：2 N/m, free resonance frequency 14 kHz), SEM (Carl Zeiss Supra 55), freezing microtome (Leica CM1950), and materials like paraformaldehyde, Tissue-Tek embedding medium, and poly-lysine. Experimental Procedures and Operational Workflow:
Samples are prepared, mounted on substrates, and characterized by SEM and AFM topography. SE-PFM is performed in lateral mode with AC voltages applied at sequential frequencies (e.g., 269-277 kHz). Data is acquired, registered, aligned, and analyzed using PCA and SHO fitting.
5：Data Analysis Methods:
PCA is applied via singular value decomposition (SVD) to extract spatial and spectral modes. SHO model fitting is used to compute intrinsic amplitude, phase, quality factor, and resonant frequency for each pixel, with goodness-of-fit assessed using R2 parameter.
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