The effective Young’s moduli of 7-4 cells measured at different indenting force at the same loading rate.

<p>Each value represents the mean ± standard error (cell number for analysis).</p

The effect of AFM tip shape, indenting force at the same loading rate, and operating temperature on the effective Young’s modulus of cells.

<p>To determine the effect of the AFM tips, (A) NIH3T3 cells and 7-4 cells were plated on type I collagen (COL I)-coated glass slides overnight. The effective Young's moduli (E_eff) of cells were measured by Bio-AFM with a pyramidal tip (labeled as P in (A)), flat tip (labeled as F in (A)), and 5 µm-bead-modified tip (labeled as B in (A)) with a 1 nN indenting force at 1 µm/sec approach velocity. To determine the effect of the indenting force at the same loading rate, cells were plated on COL I-coated glass slides overnight. The E_eff of cells were measured by Bio-AFM with a (P) pyramidal tip, (F) flat tip, and (B) 5 µm-bead-modified tip with different indenting force (0.2, 0.5 or 1 nN). To evaluate the effect of operating temperatures, (E) NIH3T3 cells were plated on COL I-coated glass slides and cultured in DMEM at 31°C, 37°C, and 43°C and in CO₂-independent medium (CO₂-IDM) at 31°C and 37°C. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. ***<i>p</i><0.001; **<i>p</i><0.01; *<i>p</i><0.05; N.S, no significance.</p

The effect of culture passage number on the effective Young’s moduli of MKPC.

<p>(A) MKPC at the 20^th, 30^th, 50^th, and 90^th passages were plated onto type I collagen- or fibronectin-coated glass slides overnight. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. (***<i>p</i><0.001; *<i>p</i><0.05) (B) MKPC at the 20^th, 30^th, 50^th, and 90^th passages were stained and represented as the maximal section of confocal immunofluorescence images of β-actin (red) and α-tubulin (green). (Scale bar = 10 µm).</p

The effect of plating density on the effective Young’s moduli of cells.

<p>(A) NIH3T3 cells and (B) MDCK cells were plated onto COL I-coated glass slides at densities of 5, 50, or 500 cells/mm² overnight. The effective Young’s moduli (E_eff) of cells were assessed by Bio-AFM. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. (***<i>p</i><0.001; N.S, no significance) (C) NIH3T3 cells were plated onto COL I-coated glass slides at densities of 5, 50, or 500 cells/mm². The immunofluorescence results are represented as F-actin (red) and nuclei (blue). (D) AFM surface topological images in living MDCK cells and confocal immunofluorescence images of F-actin (red), α-tubulin (green), and the nucleus (blue) in stained MDCK cells that were cultured at densities of 5, 50, or 500 cells/mm², respectively. (Scale bar = 10 µm).</p

The effective Young’s moduli of NIH3T3 cells measured at different indenting force at the same loading rate.

<p>Each value represents the mean ± standard error (cell number for analysis).</p

The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment

<div><p>Atomic force microscopy provides a novel technique for differentiating the mechanical properties of various cell types. Cell elasticity is abundantly used to represent the structural strength of cells in different conditions. In this study, we are interested in whether physical or physiological cues affect cell elasticity in Atomic force microscopy (AFM)-based assessments. The physical cues include the geometry of the AFM tips, the indenting force and the operating temperature of the AFM. All of these cues show a significant influence on the cell elasticity assessment. Sharp AFM tips create a two-fold increase in the value of the effective Young’s modulus (E_eff) relative to that of the blunt tips. Higher indenting force at the same loading rate generates higher estimated cell elasticity. Increasing the operation temperature of the AFM leads to decreases in the cell stiffness because the structure of actin filaments becomes disorganized. The physiological cues include the presence of fetal bovine serum or extracellular matrix-coated surfaces, the culture passage number, and the culture density. Both fetal bovine serum and the extracellular matrix are critical for cells to maintain the integrity of actin filaments and consequently exhibit higher elasticity. Unlike primary cells, mouse kidney progenitor cells can be passaged and maintain their morphology and elasticity for a very long period without a senescence phenotype. Finally, cell elasticity increases with increasing culture density only in MDCK epithelial cells. In summary, for researchers who use AFM to assess cell elasticity, our results provide basic and significant information about the suitable selection of physical and physiological cues.</p></div

The effect of various substrates on the effective Young’s moduli of cells.

<p>(A) NIH3T3 cells were plated onto glass slides coated with various substrates (PLL, poly-L-Lysine; FN, fibronectin; COL I, type I collagen; COL IV, type IV collagen; GEL; gelatin) overnight. (B) To evaluate the effect of substrate concentration on the effective Young’s moduli (E_eff) of cells, cells were plated onto glass slides coated with COL I of various concentrations (50, 100, or 1000 µg/ml). (C) To evaluate the effect of substrate compliance, cells were plated onto culture dish (C), collagen gel-coated dish (Co), and collagen gel (G). The E_eff of cells were assessed by Bio-AFM. The results were expressed as the mean ± SEM by scatter dot plot. Gray symbols represent the detailed experimental data. ***<i>p</i><0.001; **<i>p</i><0.01; N.S, no significance. (D) Organization of actin filament in the apical actin and basal actin in NIH3T3 cells plated on different substrate (glass, PLL, FN, COL I, COL IV, GEL, Co, G). (Scale bar = 10 µm).</p

The effect of culture conditions on the effective Young’s moduli of cells.

<p>NIH3T3 cells were plated onto COL I-coated glass slides and culured in DMEM (or CO₂-IDM) supplemented with or without FBS overnight. The incubating temperatures were set at room temperature (31°C), 37°C, and 43°C. (A) The cells were cultured in DMEM or CO₂-IDM supplemented with or without 10% FBS at 37°C, respectively. The effective Young’s moduli (E_eff) of cells were assessed by the Bio-AFM. The results are showed in scatter dot plot by mean with standard error (SE). **p<0.01; *p<0.05; N.S., no significance. (B) The representative Max XY projection images of cells cultured in various conditions. Actin cap fibers in the apical region of the cell were re-colored green, the stress fibers in the middle region of cell were colored red, and the stress fibers in the the basal region of cell were re-colored blue. (Scale bar = 10 µm).</p

Representative force-indentation curves from AFM and sketches of the tip geometry.

<p>(A) The dotted and solid lines represent distinct traces of the approach of the AFM tip, as measured from samples with different physical properties. Initially, the AFM tip was located at the designed position over the sample. As the AFM tip start to approach the sample, there was no interaction force (<i>Part I</i>). After the AFM tip contacted with the sample at the contact point (shown by black arrow), further indentation generates the indentation depth. Constant force generates a greater indentation depth on the softer cell (<i>Part III</i>) than on the stiffer cell (<i>Part II</i>). (B) Tips with three different geometries were used in this study.</p

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

<p>Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.</p