An Asylum MFP3D AFM (Asylum Research, Santa Barbara, CA) was used to perform the nanoindentation on single fibroblasts that were adhered to the PDMS coated substrate. The cells were indented via gold-coated polystyrene colloidal probe tips (end radius, R ~ 2.5 μm) attached to cantilivers with nominal spring constant k ~ 0.06 N/m, Novascan, Ames, IA). The thermal noise oscillation method was applied to determine the cantilever spring constant for each probe tip32 . The indentation was performed under force control scheme, with maximum force ~2.5 nN. The resulting indentation depths were in the range of 0.2–1 μm. The axial (z direction) displacement of the tip is calculated as the z-piezo subtracted from the vertical deflection of the cantilever. Typical force-displacement curves for cells in different sucrose concentrations are shown in Supplementary Fig. 7 . A typical force-displacement curve and the best-fit curve from the thin-layer Hertz model are shown in Supplementary Fig. 8 . Different locations on cells were indented to obtain an average for the Young’s modulus of each cell. An indentation speed of 0.1 μm/s was used to probe the Young’s modulus under close-to-equilibrium conditions.
Asylum mfp 3d afm
Manufactured by Oxford Instruments
Sourced in United States
The Asylum MFP-3D AFM is an atomic force microscope that provides high-resolution imaging and analysis of surface topography and properties at the nanoscale level. It utilizes a cantilever-based probe to detect and measure interactions between the sample and the tip, enabling the acquisition of detailed surface information.
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13 protocols using asylum mfp 3d afm
1
Nanoindentation of Single Fibroblasts
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Nanoindentation of Single Fibroblasts
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An Asylum MFP3D AFM (Asylum Research, Santa Barbara, CA) was used to perform the nanoindentation on single fibroblasts that were adhered to the PDMS coated substrate. The cells were indented via gold-coated polystyrene colloidal probe tips (end radius, R ~ 2.5 μm) attached to cantilivers with nominal spring constant k ~ 0.06 N/m, Novascan, Ames, IA). The thermal noise oscillation method was applied to determine the cantilever spring constant for each probe tip32 . The indentation was performed under force control scheme, with maximum force ~2.5 nN. The resulting indentation depths were in the range of 0.2–1 μm. The axial (z direction) displacement of the tip is calculated as the z-piezo subtracted from the vertical deflection of the cantilever. Typical force-displacement curves for cells in different sucrose concentrations are shown in Supplementary Fig. 7 . A typical force-displacement curve and the best-fit curve from the thin-layer Hertz model are shown in Supplementary Fig. 8 . Different locations on cells were indented to obtain an average for the Young’s modulus of each cell. An indentation speed of 0.1 μm/s was used to probe the Young’s modulus under close-to-equilibrium conditions.
3
Atomic Force Microscopy Force Curves
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Force curves were collected using an Asylum MFP-3D AFM (Asylum Research, USA) with ACTA (AppNano, USA, nominal spring constant 40 N/m) probes in air and AC240 (Olympus, Japan, nominal spring constant 2 N/m) probes in liquid. Prior to solution phase AFM experiments, samples were also soaked in 1× PBS solution at room temperature for ~2 h. Force curves were recorded by loading at a rate of 1μm/s up to an indentation depth of ~10 nm, followed by unloading at the same rate.
4
Fiber Morphology Analysis by TEM and AFM
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The morphology of fibers was assessed with Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) imaging. Bright-field TEM images were collected on a FEI Tecnai G2 F20 S/TEM operated at an accelerating voltage of 200 kV after staining the samples with uranyl-acetate or gold nanoparticles. Tapping mode (TM) AFM was performed on an Asylum MFP-3D AFM (Asylum Research, Santa Barbara, CA, USA) using Veecoprobes Sb-doped Si cantilevers (ρ = 0.01–0.025 Ω-cm, k = 40 N/m, ν~ 300 kHz).
5
Atomic Force Microscopy of Cell Cortical Stiffness
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Cells were seeded on 18mm coverslips coated with collagen and allowed to adhere for a minimum of 4 hours. AFM measurements were performed with an Asylum MFP3DAFM (Asylum Research, CA) coupled to a Nikon TE2000E2 epifluorescence microscope. Individual cells were indented using a pyramid-tipped probe (Olympus) with a nominal spring constant of 10 pN/nm. The probe was first calibrated in air using thermal vibration method to determine the exact spring constant. The first 1μm of force-indentation curves for individual cells were fitted with the Hertzian model for a pyramidal tip to obtain estimates of cortical stiffness. At least 100 cells across three experiments were analyzed per condition.
6
Fiber Morphology Analysis by TEM and AFM
7
AFM Force Measurements of Cellular Adhesion
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AFM force measurements were made using an Asylum MFP-3D AFM (Asylum Research, Santa Barbara, CA, USA) mounted on top of an Olympus IX51 inverted optical microscope for visualizing and manually positioning regions to be probed. Force measurements (force-distance curves) were made at a rate of 0.3 to 2.0 Hz, using Si3N4 or Si cantilevers modified with a glass sphere (with a radius of 10 µm), a glass sphere coated with gold layers (with a radius of 10 µm), or a polystyrene sphere (with a radius of 12.5 µm) (Novascan), with calibrated spring constants between 0.6 and 14 N/m. Both force mapping mode and continuous measurement mode methods were used in this study. All adhesion plots shown in the figures were based on data collected from force mapping mode, which were confirmed with continuous measurement mode. More detailed information about adhesion force measurements are described in Supplementary Section 15 and Supplementary Figure 21–35 .
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Atomic Force Microscopy Force Curves
9
Measuring Cellular Cortical Stiffness
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CHO cells were cultured on tissue culture plates for 24 h before initiating the experiments. Culture plates were mounted onto the stage of an Asylum MFP3D AFM (Asylum Research, CA) coupled to a Zeiss epifluorescence microscope and indented using a pyramid-tipped probe (Olympus) with nominal spring constant of 20 pN/nm. Actual spring constants were determined using thermal calibration method. Force curves were obtained for 30-40 cells for each condition and each time point. Force-indentation profiles were fit with a modified Hertzian model of a cone indenting a semi-infinite elastic material to extract the magnitude of cortical stiffness46 (link).
10
Atomic Force Microscopy Measurements
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AFM measurements were performed with an Asylum MFP3DAFM (Asylum Research, CA) coupled to an epifluorescence microscope (Nikon, Japan). Individual treated cells were indented using a pyramid-tipped probe with a nominal spring constant of 25 pN/nm. The first 1 μm of force-indentation curves was fitted with the Hertzian model for a pyramidal tip to obtain the elastic modulus. The analysis of the indentation curves was performed using MATLAB software as described previously45 (link).
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