Force Maps

Abstract of the Nature Protocols Publication:

Mammalian cells sense and react to the mechanics of their immediate microenvironment. The characterization of the biomechanical properties of tissues with high spatial resolution, therefore, provides valuable insights into a broad variety of developmental, homeostatic, and pathological processes within living organism. The biomechanical properties of the basement membrane (BM), an extracellular matrix (ECM) sub-structure measuring only ~100 - 400 nm across are, among other things, pivotal to tumour progression and metastasis formation. Although the precise assignment of the Young’s modulus E of such a thin ECM sub-structure especially in between two cell layers is still challenging, biomechanical data of the BM can provide information of eminent diagnostic potential. Here, we present a detailed protocol to quantify the elastic modulus of the BM in murine and human lung tissue, which is one of the major organs prone to metastasis. This protocol describes a streamlined workflow to determine the Young’s modulus E of the BM between the endothelial and epithelial cell layers shaping the alveolar wall in lung tissues using atomic force microscopy (AFM). Our step-by-step protocol provides instructions for murine and human lung tissue extraction, inflation of these tissues with cryogenic cutting medium, freezing and cryo-sectioning of the tissue samples, and AFM force-map recording. In addition, it guides the reader through a semi-automatic data analysis procedure to identify the pulmonary BM and extract its Young’s modulus E using an in-house tailored user-friendly AFM data analysis software, the CANTER Processing Toolbox, which enables automatic loading of the recorded force maps, conversion of the force vs. piezo-extension curves to force vs. indentation curves, calculation of Young’s moduli and generation of Young’s modulus maps, where the pulmonary BM can be identified using a semi-automatic spatial filtering tool. The entire protocol takes 1 - 2 days.