Creep Indentation of Single Cells

[+] Author and Article Information
Eugene J. Koay

Rice University, Department of Bioengineering, Houston, TX 77005Baylor College of Medicine, Houston, TX 77030

Adrian C. Shieh, Kyriacos A. Athanasiou

Rice University, Department of Bioengineering, Houston, TX 77005

J Biomech Eng 125(3), 334-341 (Jun 10, 2003) (8 pages) doi:10.1115/1.1572517 History: Received March 14, 2002; Revised December 18, 2002; Online June 10, 2003
Copyright © 2003 by ASME
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Setup of Creep Cytoindentation Apparatus. (a) A photograph of the Creep Cytoindentation Apparatus (CCA), showing the major components of the system arranged for a typical experiment. (b) The CCA utilizes a closed loop control algorithm to apply a constant stress to the surface of anchorage dependent cells. It consists of a computer with data acquisition hardware and software (A), a controller (B) for the piezoelectric translator (C), a 75 μm diameter glass cantilever and 5 μm diameter glass probe (D), a laser micrometer (E), and an active tunable filter (F); other components include a manual positioning device for the piezoelectric translator (which is connected to the cantilever) (1), an inverted microscope (2), and a manual positioning device for the laser (3). The boxed area is enlarged in Fig. 5.
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Deflection of cantilever. Calculating the deformation of the cell first required knowledge of the deflection at the point of contact (y1). This was determined with the experimentally measured deflection (δ) and other constants (Young’s modulus (E), moment of inertia (I),L1, and L2). The initial position of the probe at the application of the test load and the deflection at the point of contact allowed the deformation of the cell to be determined.
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Calibration. The piezoelectric translator controls the base of the cantilever while the laser micrometer records the displacement of the end of the cantilever. Due to refraction by the quartz window and growth media, a calibration procedure is conducted whereby the piezoelectric translator moves a known distance and the laser data is recorded. By dividing the known movement by the recorded laser data, a refraction constant is obtained. The piezoelectric translator is held for a few seconds to test the stability of the laser signal as well.
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Validation Setup. A cantilever beam, designated ‘Test Beam,’ with known Young’s Modulus (Et), moment of inertia (It), and length (Lt) was used as resistance to the cantilever beam used in experiments, designated ‘Cantilever Beam’ in the figure. By measuring the deflection of ‘Cantilever Beam,’ an experimentally derived value for the Young’s Modulus of ‘Test Beam’ could be compared to the factory provided value, offering a method to validate the apparatus in terms of force and displacement measurements. This procedure was conducted in similar conditions as those during actual experiments on cells.
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Experimental Setup. (a) Fixed 1 mm from the end of the 75 μm diameter glass cantilever is the 5 μm diameter glass probe. A piezoelectric translator is used to control the movement at the base of the cantilever, while a laser micrometer records the displacement of the end of the cantilever. The laser readings are facilitated by a reflective chrome coating on the end of the cantilever and by a quartz window (not shown). (b) A photograph of a bovine articular chondrocyte seeded for 3 hrs on a glass coverslip. The round morphology of the cell is typical for 1st passage chondrocytes cultured under these conditions (400× magnification).
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Force Profile. In this particular force profile, a tare load of 5 nN and test load of 50 nN were used. Individual points obtained from experiments are shown with the desired force profile outlined. Times for these loads and the values of the tare load were varied slightly to establish a functional relationship. While this force is applied, the corresponding deformation of the cell is recorded and can be seen in Fig. 7. During indentation, the area in contact with the cell surface is constant, allowing stress-controlled experiments to be conducted.
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Standard Linear Solid Curve Fit. The standard linear solid model is a three parameter mathematical model that accounts for the time dependent viscoelastic behavior of the cell. The figure demonstrates the instantaneous deformation that occurs after the test load is applied. The rate of deformation reaches equilibrium on the order of seconds.




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