Optimization of sample preparation methods for the mechanical characterization of tissue architectures

Abstract

There is now mounting evidence that mechanical signals are as crucial as genetic and biochemical feedback mechanisms for directing and organizing complex cellular behaviors, and material characterization tools are routinely being employed in biomedical research to investigate the physical aspects of cell-to-cell communication. Atomic force microscopy (AFM) is a surface characterization tool that is compatible with aqueous environments and has recently emerged as an important technique for the mechanical analysis of biomaterials such as proteins, nucleic acids, cells and tissues. However, owing to the natural heterogeneity of biological materials and the diversity of sample preparation methods, AFM results in the literature are characterized by large discrepancies between individual studies, which prevents the drawing of general conclusions from the existing research. While the effects of factors such as AFM probe morphology and fixation length on measurement quality have been described individually in the literature, previous studies typically focus on the analysis of bacterial and eukaryotic cells rather than tissues. Consequently, a detailed comparison of tissue preparation methods for AFM analysis is lacking. The present thesis describes the mechanical characteristics of four murine tissues (heart, liver, spleen and kidney) following sample preparation using three commonly employed methods (paraffin-embedding, cryosectioning and agarose-embedding). Fixatives used in the paraffin-embedding process are observed to greatly increase the elastic modulus of tissues due to the irreversible cross-linking of the tissue extracellular matrix. Cryosectioning and agarose-embedding, in contrast, provide elasticity values that are more consistent with the live condition of the tissue, but suffer from high tip-sample adhesion that must be compensated through the use of high (~10 μm) measurement distances and/or non-adhesive AFM probes. In addition, agarose-embedded sections are subject to stringent measurement conditions due to the lack of long-term storage options. While cryosectioning is found to be an ideal compromise between data quality and ease of measurement, perfusion with 4% PFA is observed to increase tissue elasticity even under cryosectioning, showing that perfusion is not a recommended step in tissue preparation for AFM.