Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Abstract

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young's (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

Regulators, functions, and mechanotransduction pathways of matrix stiffness in hepatic disease

“…There is an equation between elastic modulus and shear modulus, and the elastic modulus is approximately three times the shear modulus for isotropic and incompressible materials. Several mechanical test methods have been used to measure the matrix stiffness, including elastography and atomic force microscopy (AFM) methodology (Sigrist et al, 2017;Ojha et al, 2022).…”

“…One of the major drawbacks of common liver stiffness measurements including USE and MRE is that they do not provide cellular-level resolution of stiffness heterogeneity in the liver (Ojha et al, 2022). During the liver disease development, areas containing rich collagens show higher stiffness compared to the surrounding parenchyma (Calò et al, 2020).…”

Recent progress of second near-infrared (NIR-II) fluorescence microscopy in bioimaging

“…For example, microscopy is useful for observing the transfer of the human immunodeficiency virus (HIV) (Hubner et al, 2009) from cell to cell, the separation of individual chromatids during cell division, the transient activity of the entire brain of zebrafish larvae, and the neuronal activity of entire worms (Gao et al, 2012;Hontani et al, 2022;Ishii et al, 2022). In vivo imaging studies through macro NIR-II imaging can not only detect circulation in the aorta and tiny blood vessels, but also be used for the imaging of various organs, such as the heart (Greiner et al, 2022), liver (Ojha et al, 2022), lung (Mirsanaye et al, 2022), kidney (Sonoda et al, 2022) and intestine (Hanafy et al, 2022). However, the visualization of tissue microstructure requires imaging systems with larger magnification for improved spatial resolution and contrast of biological tissue and clear imaging of biological microstructure.…”