Jpk nanowizard 4

Surfaces were imaged in tapping
mode (JPK NanoWizard 4, Bruker Nano GmbH, USA) with a cantilever (type
OPUS, 160-AC-NA, back side coating with reflective aluminum) with
300 kHz resonance frequency, a spring constant of 26 Nm^–1 and a nominal tip radius <7 nm (Figure S3). The calculation of the root mean square roughness (RMS) roughness
was done with the software Gwyddion on part of the images of 15 ×
15 μm². The images were leveled using a polynomial
background of first degree (offset and plane). Each process was repeated
with three independent samples. Each sample was measured in at least
two different positions. The standard error was calculated using the
formula SE = σ × n^–1/2, where σ
represents the standard deviation and n represents the sample size.
The measured root-mean-square roughness of the surfaces is summarized
in Table 1.

AFM imaging was performed at room temperature on a JPK NanoWizard 4 (Bruker, MA, USA) operated in tapping mode and using MSNL cantilevers (Bruker, MA, USA). Open source Gwyddion software (version 2.60, http://gwyddion.net/) was used for image processing [37 (link)].

Dry microgel films were imaged via AFM to determine the microgel morphology. The films were prepared by drying a 0.01 mg mL⁻¹ microgel dispersion on glass coverslips followed by immersing in water and drying under a stream of nitrogen. For AFM imaging, the JPK NanoWizard 2 (JPK Instruments AG, Berlin, Germany) with cantilevers with a nominal spring constant of 40 N m⁻¹ (HQ:XSC11/NO AL, MikroMash, Bulgaria) was used in tapping mode. For elastic modulus determination, a JPK NanoWizard 4 (Bruker Nano GmbH, Berlin, Germany) in the QI (quantitative imaging) mode, with cantilevers with a nominal spring constant of 2.7 N m⁻¹ (HQ:XSC11/NO AL, MikroMash, Sofia, Bulgaria) with a nominal tip radius of 8 nm, was used. The measurements were conducted in lectin binding buffer (LBB, 10 mM HEPES, 1 mM CaCl₂, 1 mM MnCl₂, pH 7.4) using the microgel coatings as described above.

For the in vitro surface area analysis, Roxolid discs were prepared with a modified-SLA-like surface (mod-dry/micro) and a SLActive surface, accelerated aged and then air dried under a laminar flow hood (dry/nano). Atomic force microscopy (AFM) is the most suitable technique for measuring nanoscale surface topography of hard surfaces, but the sandblasting process leads to undesirable artefacts in the measurement process; the sandblasting step was therefore omitted without impacting the low-micro and nanotopography, which are a result of the etching and aging process.
AFM measurements were performed using a JPK Nanowizard 4 (Bruker Nano GmbH, Berlin, Germany) with a direct drive cantilever holder and SSS-NCHR cantilevers with a tip radius of 2–3 nm (Nanotools GmbH, Munich, Germany) in dynamic mode. An area of 2 × 2 µm² was scanned for every measurement at 0.25 Hz/line and recorded using 2048 × 2048 points. The data were analyzed using µSoft Analysis XT software (version 5.1.1.5944; NanoFocus AG, Oberhausen, Germany) according to ISO 25,178 using a Gaussian filter with a cut-off wavelength of 0.25 µm and the area ratio between mod-dry/micro and dry/nano was estimated as (SdrmodMA/100 + 1)/(SdrMA/100 + 1), where Sdr is the mean developed interfacial area ratio. For each sample group, 5 discs were measured at two random positions.

To investigate the protein coverage and protein height, atomic force micrographs of the SLB surface before and after protein addition were taken. ENTH^WT and ENTH^R114A (1 µM) were added for 2 h at RT. The solution was mixed with a stirring bar to ensure homogenous distribution of the protein. Afterwards 10 × 10 µm² and 1 × 1 µm² areas of the substrates were imaged in contact mode with BL-AC40TS-C2 cantilevers (f = 85.4–139.1 kHz, k = 0.03–0.12 N m⁻¹, Olympus, Tokio, Japan). Measurements were performed using a JPK Nanowizard 4 (JPK Instruments, Berlin, Germany) equipped with a CCD camera (Fire Wire CCD color camera, The Imaging Source, NC, USA). The protein height and cluster surface coverage were calculated with a MATLAB routine.