Nanowizard 2 afm system

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.

Cells were plated at the density of 3×10³ cells/cm², exposed to OIM for 48 h, and the stiffness of a cell was measured with the BioCell device (JPK Instruments, Berlin, Germany) (Chiou et al., 2013 (link)) on the JPK NanoWizard II AFM system (Costa, 2004 (link); Li et al., 2008 (link)).

For AFM analysis, 20 μl of three independent bacterial cultivations were spotted onto glass coverslips (Hartenstein). After 1 h, samples were rinsed with ultrapure water to remove media residues before mounted onto microscopy slides for immediate AFM imaging.
AFM measurements were conducted under ambient conditions using the NanoWizard II AFM system (JPK Instruments AG, Berlin, Germany) by driving the AFM in soft contact mode using silicon nitride AFM probes with a nominal force constant of 0.06 N/m (SiNi, Budget Sensors, Wetzlar, Germany). Scan rates were set to 1 Hz and images were acquired with a resolution of 512 × 512 pixels. For each sample, topographic overview images were taken before zoom-ins on cells or appendages were performed. Representative height and deflection images are displayed in false-color. All height images were XY tilt corrected, polynomial- fitted and unsharpened mask filtered to remove noise using JPK data processing software (JPK Instruments AG). Height dimensions of fimbriae were determined after XY tilt correction from raw images and presented as mean values (n = 20). The positions for the analysis were carefully chosen to ensure that individual fimbria rather than bundles were measured. The values were derived from Z-dimensions since X-and Y-measurements are affected by the tip geometry.