Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods  Alexander Cartagena, Arvind Raman 

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Date of download: 12/28/2017 Copyright © ASME. All rights reserved.
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Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods  Alexander Cartagena, Arvind Raman  Biophysical Journal  Volume 106, Issue 5, Pages 1033-1043 (March 2014) DOI: 10.1016/j.bpj.2013.12.037 Copyright © 2014 Biophysical Society Terms and Conditions

Figure 1 (a) Schematic of a Lorentz force excited microcantilever interacting with a live fibroblast cell by dynamic spectroscopy. In conventional quasi-static spectroscopy a static cantilever is ramped toward the soft cell and retracted to record an F-Z curve. However, in dynamic spectroscopy a microcantilever is excited at near the resonant frequency and moved toward the sample and then retracted to record three channels of information: mean deflection (A0), first harmonic amplitude, and phase lag (A1 and ϕ1). (b) Tune curves performed at 4 μm away and in contact with the cell with a force of ∼1 nN show that the transfer functions of a fully vibrated cantilever changes when interacting with the cell, resulting in changes in amplitude and phase that are closely related to the cell viscoelastic properties. Therefore, an equivalent spring-dashpot model for the cantilever in contact with the cell can be used to extract the viscoelastic properties. To see this figure in color, go online. Biophysical Journal 2014 106, 1033-1043DOI: (10.1016/j.bpj.2013.12.037) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 2 Dynamic approach curves performed on different locations across the sample. (a) Three-dimensionally rendered topography image of a live fibroblasts. (Crosses) Measurement locations. (b–d) Dynamic-approach curves for multi-harmonic observables (A0, A1, and ϕ1) acquired in the nucleus and periphery of a live fibroblast cell and the stiffer gelatin-coated petri dish, respectively. (Green vertical lines) Typical imaging setpoint ratios of 85 and 75%. (Curves) Behavior of the multi-harmonic amplitudes and phase (A0, A1, and ϕ1) as the microcantilever moves toward and interacts with different regions of a live fibroblast cell in a soft repulsive regime. It is clear that the strongest and most easily accessible harmonic signals that reflect local viscoelastic properties are those from the 0th and 1st harmonics for live cells. Topography image was acquired using a Lorentz force excited Olympus (Melville, NY) microcantilever (BL-TR400PB: kcant = 0.083 N/m, Q = 1.9, ωdr = 7.79 kHz, and A1far = 8.5 nm). To see this figure in color, go online. Biophysical Journal 2014 106, 1033-1043DOI: (10.1016/j.bpj.2013.12.037) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 3 Frequency- and indentation-depth dependence of the viscoelastic properties measured on a live fibroblast cell. The dynamic viscoelastic properties have been measured at a fixed high excitation frequency of ωdr = 7.79 kHz. (a and b) Dynamic ksampledynamic (blue) and static ksamplestatic (red) force gradients at different mean indentations δ0 on the nuclear and peripheral region of a live fibroblast cell. Increase of ksampledynamic/ksamplestatic force gradients after a critical indentation depth is observed in the cell nucleus but not in the periphery. (c) Damping csampledynamic at different mean indentations δ0 on the nucleus and periphery region of the cell. The plots are the averages of 10 dynamic and static approach curves performed in each location on the cell. (Insets) Images are the topography of the cell probed. (Crosses) Area where the measurements were made. To see this figure in color, go online. Biophysical Journal 2014 106, 1033-1043DOI: (10.1016/j.bpj.2013.12.037) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 4 Viscoelastic properties response for different fibroblast cells. (a) Dynamic ksampledynamic (solid lines) and static ksamplestatic (dashed lines) force gradients and (b) damping csampledynamic as a function of mean indentations δ0 on the nuclear region. (c and d) Same measurements are collected on the peripheral region. The plots represent the averages of 10 dynamic and static approach curves performed in each specified location on each cell. Increase of ksampledynamic/ksamplestatic force gradients in the nucleus is observed for every cell, which confirm our major finding that there are difference in viscoelastic properties in the nucleus and is initially frequency-dependent, but after a critical indentation depth is frequency- and indentation-dependent. The dynamic viscoelastic properties of the cells have been measured using three different cantilevers at a fixed high excitation frequency ωdr and with kcant and Q. Cell No. 1: 7.79 kHz, 0.083 N/m, and 1.9. Cell No. 2: 7.69 kHz, 0.076 N/m, and 1.8. Cell No. 3: 8.18 kHz, 0.076 N/m, and 1.8. (Insets) Topography images of the cells. (Crosses) Locations where the measurements were made. To see this figure in color, go online. Biophysical Journal 2014 106, 1033-1043DOI: (10.1016/j.bpj.2013.12.037) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 5 Multi-harmonic AFM images acquired on a live fibroblast cell in physiological conditions. (a) Optical phase contrast micrograph using a 10× objective; (b) topographic image; (c) 0th harmonic amplitude map A0; (d) first harmonic phase map ϕ1; (e) applied mean normal force map F0ts; and (f–h) maps of local dynamic force gradient ksampledynamic, damping csampledynamic, and loss tangent tanδ, respectively, extracted from the measured 0th and first harmonic data using the theory described in the text. Surprisingly, the loss tangent values of the cell periphery and dish are larger than the cell nucleus. Images were acquired using a Lorentz force excited Olympus (Melville, NY) microcantilever (BL-TR400PB: kcant = 0.083 N/m, Q =1.9, and ωdr = 7.79 kHz). The scale bar on images represents 10 μm (size; 60 × 60 μm2, pixels; 256 × 256). To see this figure in color, go online. Biophysical Journal 2014 106, 1033-1043DOI: (10.1016/j.bpj.2013.12.037) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 6 Plots are the averages of 10 dynamic force gradients of (a) ksampledynamic and (b) damping csampledynamic against the applied force performed on the nucleus (blue), periphery (red) of the live fibroblast cell, and the gelatin-coated petri dish (green). At forces higher than ∼800 pN on the cell nucleus the dynamic force gradient and damping becomes higher than the periphery and dish, increasing rapidly as the cell indentation progressively increases. This explains the stiffness and damping contrast between the peripheral and nuclear regions observed in Fig. 5’s maps. To see this figure in color, go online. Biophysical Journal 2014 106, 1033-1043DOI: (10.1016/j.bpj.2013.12.037) Copyright © 2014 Biophysical Society Terms and Conditions