Otespa r3

Atomic force
microscopy was
conducted in dry state with a Bruker Dimension FastScan BioTM atomic
force microscope, which was operated in Tapping mode. AFM probes with
a nominal force constant of 26 N/m and resonance frequency of 300
kHz (OTESPA-R3, Bruker) were used. Samples were scanned with scan
rates between 0.6 and 1 Hz. Images were processed with NanoScope Analysis
1.8.

Dimensions of polymer nanostructures were measured by AFM in intermittent-contact mode using a Bruker Dimension 6100 (Santa Barbara, CA) instrument. Images were collected on freshly cleaved mica and graphite surfaces. Upon cleavage, surfaces were exposed to polymer solutions for 10 min and then rinsed with DI H₂O to remove any salt formations. Imaging was performed using rectangular probes with 5–10 nm wide tip apex (OTESPA-R3, 26 N/m, 300 kHz, Bruker, Camarillo, CA) at a scan rate of 0.5 Hz. Images ranged from 512 to 1024 pixels in both fast and slow axis. End- and mid-points for nanorod length calculations were collected using Gwyddion v2.37 (Czech Metrology Institute, Czech Republic) software. Nanorods selected for rigidity analysis were clearly not in contact any other nanorods, and were longer than 50 nm.

Atomic force microscopy (AFM) was performed on a Dimension Edge AFM (Bruker S.A.S., France) in tapping mode. OTESPA-R3 (Bruker S.A.S., France) cantilevers with a spring constant of ~26 N/m were used for imaging. AFM image analysis was performed in Gwyddion v2.49. Mainly, the correction algorithms “remove polynomial background,” “align rows using various methods,” and “correct horizontal scars (strokes)” were applied for leveling. Background subtraction was performed by taking the signal of the glass coverslip not covered by protein as a reference. For quality assessment of protein transfer by printing, AFM images were converted to 16 Bit grayscale images and further analyzed in ImageJ. For this, regular arrays were selected within the printed patterns (Figure S1) corresponding to either regions with (“ON”) or without (“OFF”) stamp-surface contact. The mean gray values per pixel for each region, I_ON and I_OFF, were used to calculate the contrast $C=\frac{I_{ON}-I_{OFF}}{I_{ON}}$ . In AFM image analysis, care was taken to perform the background subtraction the same way in all images to make contrast data comparable. Note though that contrast values are only intended as a means of relative comparison between samples and are no absolute indicator of pattern quality.

AFM experiments were carried out using a JPK NanoWizard IV microscope (Bruker, Billerica, MA, USA). Tapping mode AFM imaging was performed using Si cantilevers (OMCL-AC160TS, Olympus, Tokyo, Japan) with a typical resonant frequency of ∼300 kHz and a nominal tip radius of ∼7 nm. To evaluate the aminophosphonate layer thickness, we performed nanoshaving experiments by scanning a selected area in hard contact mode to selectively displace molecules and obtain an exposed substrate region. Shaving experiments were performed using Si₃N₄ cantilevers (OTESPA-R3, Bruker) with an elastic constant 26 N/m. Typical forces applied for shaving were in the range (200 ÷ 400) nN. After shaving, images with larger scan size were acquired in tapping mode. Data were analyzed with Gwyddion (v2.55) and JPKSPM Data Processing software (v7.0.162).

The AFM is a Veeco Nanoman V (Bruker, MA, USA) equipped with an accurate three-axis scanner operating under closed-loop control (hybrid XYZ-scanner with a range of 90 μm × 90 μm × 8 μm). All measurements were carried out with OTESPA-R3 (Bruker, MA, USA) probes (roughly 7 nm nominal radius of curvature of the tip) using the tapping mode. The nominal stiffness of the cantilever is 42 N/m and its resonance frequency is 300 kHz. The tip oscillation amplitude was always about 40 nm. The amplitude setpoint was set near the free amplitude (80%) value to prevent too strong interactions with the sample nanoparticles which could lead to their displacements. On all images, the pixel size was close to 5.0 nm. The scan speed was kept to 4 μm/s with constant feedback parameters (0.8 for integral gain and 10 for the proportional one).

Atomic force microscopy (AFM) imaging was performed in tapping mode using a Nanoscope IIIa-MultiMode AFM (Digital Instruments, Santa Barbara, CA) equipped with the E (10 μm) scanner. We used silicon cantilevers (Bruker OTESPA-R3), with nominal resonance frequency 300 kHz and nominal tip radius of 7 nm.
Height and length distribution where obtained with ImageJ and the Ridge Detection plugin (https://imagej.net/Ridge_Detection) based on the algorithm for detecting ridges and lines described by Steger^{47 (link)–49} . The height and length histograms for d(G₂AG₄AG₂) G-wires were obtained by detecting all G-wires (N = 907) in eight AFM images (Supplementary Fig. 1). The height and length histograms for d(G₂AG₄CG₂) G-wires were obtained by taking into account all detected G-wires (N = 3766) in 12 AFM images (Supplementary Fig. 24a). In case of (G₂AG₄CG₂) G-wires deposited on mica, which was not pre-treated with saturated solution of MgCl₂, the height and length histograms were obtained by taking into account all detected G-wires (N = 2646) in 20 AFM images (Supplementary Fig. 24b). One should note that the actual G-wire lengths are around 10 nm shorter than what is observed by AFM due to the finite tip size effect⁵⁰ .

To measure the morphology of RBCs on dried smears, we used an MFP-3D AFM (Asylum Research, Oxford Instruments) as in Ref.^{16 (link)}, which was operated in AC mode at a scanning speed of 0.25 Hz. Each scan was performed on an area of 90 $\times$ 90  $\mathrm{\mu }$ m with a resolution of 512 $\times$ 512 pixels. The cantilever was an OTESPA-R3 from Bruker with a rectangular shape, a tip radius of 7–10 nm, a spring constant of 26 N/m, operated at a frequency of 280–300 kHz with a drive amplitude of 250–300  mV. The software used for the set up and acquisition of the AFM was Igor Pro 6.37. Fig. 8 shows representative images of RBCs with artefacts due to tip contamination. Despite the bad quality of the image, these cells were classified correctly.Figure 8

Artefacts in AFM images due to tip contamination. (a–d) Representative images for each cell category. All images were classified correctly by the neural network, showing its insensitivity to local variations in height.