Jpk data processing software

Isolated biosilica was immobilized on a poly-l-lysine-coated glass specimen holder by incubation for 1 h at room temperature. The AFM measurements were carried out on a Nanowizard IV (JPK Instruments) utilizing the QI^TM measurement mode with a trigger force of 200 pN. Biolever Mini (BL-AC40-TS, Olympus Micro) was used as cantilevers for the measurements. Calibration of the cantilevers was done by the contact-free method according to the manufacturer’s instructions. Image correction and flattening were performed within the JPK data processing software (JPK Instruments). The height of the costae was calculated using the “Extract profile” tool of the program Gwyddion. For this purpose, a line of at least 100 nm was drawn on top of a costa and in the valley next to this costa to calculate their average height profile. The net height of a costa was calculated by subtracting the average height of the valley from the average height of its neighboring costa.

Acquisition of adhesion data was performed using a NanoWizard II AFM (JPK Instruments, UK) employing a CellHesion module (JPK Instruments, UK), operating in force spectroscopy mode at 18°C. The sample and cantilever were immersed in acidic and alkaline aqueous solutions, contained within a clean glass Petri dish. The solutions were manufactured using dH₂O and pH adjusted with NaOH 0.01 M or HCl 0.01 M, to achieve solutions of pH 3, 5, 7 and 9. The samples were immobilised using a double-sided Shintron adhesive tape (Agar Scientific, UK).
The vertical deflection and z-axis displacement data were recorded at a frequency of 10 kHz. A grid of 100 force-displacement curves was acquired for each sample/liquid combination, equally spaced over an area of 100 μm × 100 μm. The force-displacement data were analysed using JPK Data Processing software (JPK Instruments, UK).
A schematic of the measured forces is shown in Fig. 1. The out-of-contact repulsion force (F_rep) was measured in the first peak from right to left of the approaching curve, the height of the triangle that was formed in the peak from the origin to the highest part of the peak until the signal was stable in the approach curve was measured as the force. The jump to force (F_JT) was measured in the minimum point of the approach curve, and the pull-off force (F_PO) was measured in the minimum point of the retract curve.

Single-cell topographical and mechanical characteristics of living CAL27 cells were measured by a AFM nano-indentation method (JPK Instruments AG, Germany), as described previously (26 (link), 27 (link)). Adhesion energy in the cytoplasmic regions was obtained using the JPK data processing software (Version spm-4.2.50, JPK, Germany) (28 (link), 29 (link)).

ImageJ software (NIH, Bethesda, MD, USA) was applied for STED image analysis. AFM images and force–distance curves were analyzed with the JPK data processing software (JPK Instruments). For unbinding position and binding strength values per experiment, the mean of two median values from all unbinding events per region of interest was applied. For image composition and processing the Photoline software (Computerinsel, Bad Gögging, Germany) was used. Densitometric measurements were done with ImageJ software (NIH, Bethesda, MD, USA). Further data shown in here were evaluated and depicted with Microsoft Excel (Microsoft, Redmond, WA, USA), RStudio (Boston, Massachusetts, USA) and Prism Software version 8 (Graph Pad, San Diego, California, USA).

Cell surface stiffness was examined by indentation assay using an AFM cantilever as described previously^{55 (link), 56 (link)}. Briefly, force probe was prepared by attaching a glass bead (ca. 100 μm diameter) to a tipless silicon cantilever (450 μm long, 50 μm wide, 2 μm thick; nominal spring constant 0.02 N/m; TL-CONT, Nanosensors) using two component Araldite epoxy glue. The cells were set on a plastic culture dish, and pressed from the apical side with a force of 20 nN (the approach and retraction speeds were set to 1.0 μm/s) by the force probe. The measurement using a contact mode was carried out with the Nano-Wizard system with the Cell-Hesion module (JPK), and the data analysis was done with the JPK data processing software (JPK Instruments), where the cell elasticity (Young’s modulus) in the apical region was estimated from a force–distance curve using the Hertz model. The cells were cultured in Leibovitz’s L-15 medium (Thermo Fisher). Nocodazole treatments were given for 60 min at 37 °C before the measurements. All measurements were performed at room temperature.

AFM measurements were conducted using the NanoWizard II AFM system (JPK Instruments AG, Berlin, Germany). High-resolution surface images were acquired by operating the AFM under ambient conditions in soft contact mode using silicon nitride AFM probes with a nominal force constant of 0.06 N/m (SiNi, Budget Sensors, Wetzlar, Germany). Samples were prepared as described above. For each sample, topographic overview images with a 90 x 90 µm scan area were taken before zoom-ins were generated. All images were polynomial fitted and unsharpened mask filtered using JPK data processing software (JPK Instruments AG). 3D projections of height profiles are shown, tilted 12° in X direction.
To correlate surface structures recorded by AFM with the cytoskeleton, epifluorescence images of Lifeact-eGFP cells were acquired and aligned with AFM images by matching landmarks observed in both images using the transform tool in Adobe Photoshop.

For image processing, Adobe Photoshop CS5 (Adobe, Dublin, Ireland) was used. AFM images and data analysis of force–distance curves were done on JPK Data Processing Software (JPK Instruments). For further calculation of the analyzed AFM data with regard to unbinding forces, peak fitting, and step position Origin Pro 2016, 93G (Northampton, MA, USA) was used. In addition, Origin was utilized for comparison of data values with a paired Student’s t-test (two sample groups) or one-way analysis of variance following Bonferroni correction (more than two groups), respectively. Error bars given in the figures are mean ± SD for all diagrams depicting unbinding forces ± SEM for all other experiments. Significance was presumed at a p-value < 0.05.