Single-Cell Elastography: Probing for Disease with the Atomic Force Microscope

139 Disease Markers 19 (2003,2004) 139–154 IOS Press Single-cell elastography: Probing for disease with the atomic force microscope Kevin D. Costa∗ Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA Abstract. The atomic force microscope (AFM) is emerging as a powerful tool in cell biology. Originally developed for highresolution imaging purposes, the AFM also has unique capabilities as a nano-indenter to probe the dynamic viscoelastic material properties of living cells in culture. In particular, AFM elastography combines imaging and indentation modalities to map the spatial distribution of cell mechanical properties, which in turn reflect the structure and function of the underlying cytoskeleton. Such measurements have contributed to our understanding of cell mechanics and cell biology and appear to be sensitive to the presence of disease in individual cells. This chapter provides a background on the principles and practice of AFM elastography and reviews the literature comparing cell mechanics in normal and diseased states, making a case for the use of such measurements as disease markers. Emphasis is placed on the need for more comprehensive and detailed quantification of cell biomechanical properties beyond the current standard methods of analysis. A number of technical and practical hurdles have yet to be overcome before the method can be of clinical use. However, the future holds great promise for AFM elastography of living cells to provide novel biomechanical markers that will enhance the detection, diagnosis, and treatment of disease. Keywords: Cell biomechanics, viscoelasticity, cytoskeleton, indentation, stiffness, disease markers 1. Introduction Many physiologic and pathophysiologic processes alter the biomechanical properties of the tissues they affect. It is well known that muscles get harder with weight training, and skin becomes less resilient with age. Abnormal tissue biomechanics also play a key role in a wide range of diseases such as osteoporosis, osteoarthritis, cystic fibrosis, muscular dystrophy, ventricular aneurysm, and others. Based on the relationship between tissue mechanics and pathology, palpation is used clinically to detect stiff nodules associated with breast cancer and abdominal hardness due to cirrhosis of the liver. In an effort to make such examinations more quantitative, a number of indentation devices have been developed to evaluate the stiffness ∗ Address for correspondence: Kevin D. Costa, Ph.D., Columbia University, Department of Biomedical Engineering, 351 Engineering Terrace, Mail Code 8904, 1210 Amsterdam Avenue, New York, NY 10027, USA. Tel.: +1 212 854 9163; Fax: +1 212 854 8725; E-mail: of soft tissues in vivo [1–4], though these have yet to achieve wide clinical acceptance. Recently, there has been great interest in a new technique known as elastography [5], which generally refers to any imaging modality that yields information about the mechanical properties of a tissue. Based primarily on ultrasound and magnetic resonance imaging methods, elastographic techniques have demonstrated the ability to detect the size and shape of tumors [5, 6], to identify regional anatomic differences in normal tissue stiffness [5,6], to identify abnormal cardiac deformation due to coronary artery disease [7,8], and even have been implemented in a catheter system for intravascular evaluation of atherosclerotic plaques [9]. However, in an elastogram, image contrast is based on regional differences in the response of tissue structures to applied loads, yielding new information not available using traditional medical imaging modalities. Consequently, there is rapidly growing clinical interest in the ability to diagnose disease based on analysis and visualization of regional tissue mechanical properties. It follows that pathophysiologic changes in the mechanical properties of tissues may be manifest at the ISSN 0278-0240/03,04/$17.00  2003,2004 – IOS Press and the authors. All rights reserved K.D. Costa / Single-cell elastography: Probing for disease with the atomic force microscope single cell level. In fact, alterations of cell mechanical properties recently have been reported in certain forms of cancer, arthritis, and cardiovascular disease [10– 13], opening a new window to examine the underlying mechanisms of these pathologies. Moreover, once the normal and abnormal mechanical properties of a given cell type are established, it is enticing to imagine that potential pharmaceutical or genetic treatments might be evaluated by measuring their effects on the mechanical properties of target cells in vitro. Hence, by complementing other evolving single-cell analysis techniques [14,15], the identification of a distinct biomechanical fingerprint of the cell in response to a battery of material tests may offer an important new approach in cell biology. Single cell elastography using atomic force microscopy is a technique with the potential to identify such a mechanical fingerprint. At present, atomic force microscope (AFM) elastography is largely a research tool used by biomedical engineers and biophysicists to study the mechanics of cell function. However, the technique is evolving rapidly to a state where medical applications may be feasible. Therefore, the purpose of this article is to provide a brief introduction to cell biomechanics and its relation to disease; to describe the AFM experiment, including principles of operation and methods of data analysis; to review recent findings in the area of cell mechanics with AFM; and to identify the current limits of the technology and future developments that would enhance transfer to the basic and clinical sciences to aid in the identification of novel cell biomechanical markers that might lead to improved detection, diagnosis, and treatment of disease. 1.1. Basic cell biomechanics Such a detailed characterization of cell mechanics requires knowledge of the constitutive relation of the cell, which relates cell deformation (i.e., strain) to internal forces and externally applied loads (i.e., stress) acting on the cell. Stiffness is defined as the slope of the force-deformation curve – it depends on geometry and hence on the particular sample studied and the testing device used. Therefore, rather than relating force (F ) and deformation (∆L) directly, it is important to consider the related quantities stress (σ = F/A) and strain (ε = (∆L/Lo ) because these are normalized measures (by area, A, and initial length, L o , respectively) independent of size or geometry. That is, the stress-strain constitutive relation reflects an underlying property of the cell. Perhaps the best known and sim- stress, σ 140 ing load g din a nlo u strain, ε Fig. 1. An idealized linear elastic material (dotted line) is characterized by the Young’s modulus obtained from the slope of the stress-strain curve. For most biological soft tissues, the stress-strain relation is nonlinear (...truncated)