Nanowizard 2 afm

SCFS measurements were performed using a NanoWizard II AFM (JPK Instruments, Berlin, Germany) mounted on top of an Axiovert 200 inverted microscope (Carl Zeiss, Jena, Germany). For the cell mechanical properties measurements, standard pyramidal tipped cantilevers OMCLTR 400PSA HW (Olympus) had a nominal spring constant of 0.02 N/m. The cantilevers were calibrated by the thermal noise method before each experiment [30 (link)]. Cells were cultured on glass coverslips (ϕ 24 mm) for 24 h before the experiment. All experiments were performed at 37 °C using a temperature-controlled BioCell chamber JPK Instruments (Berlin, Germany). Force-distance curves were collected with a force load of 0.4 nN and at a rate of 2.5 μm/s. Measurements were always performed over the nuclear region of the cells. Five curves were acquired for every cell, and in every experiment, a minimum of 30 cells was analyzed. Force-distance curves were analyzed with JPK data processing software. Cell mechanical properties were acquired by evaluating Young’s modulus (E) of the cell, applying the Hertz-Sneddon model [31 (link)].

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.

Particle size, zeta potential, and size distributions of GO and its related compounds
were analyzed by photon correlation spectroscopy using a Zetasizer instrument (ZEN3600,
Nanoseries, Malvern, UK). Shape and surface morphology was analyzed using atomic force
microscopy (AFM). For AFM analysis, 20 µl of the sample was placed on mica slide,
air-dried, and visualized under the Nanowizard II AFM (JPK Instruments, Germany)
microscope. The images were acquired in contact mode under ambient conditions. The
chemical composition and the surface functional groups were analyzed by Fourier transform
infrared spectroscopy (FTIR, Thermo-Nicolet spectrometer) with KBr pellets in the range of
400–4000 cm⁻¹. UV-Vis spectrometer (Hitachi Inc., U-2910) was used to measure
the optical absorption properties of GO and related products.

Young’s moduli of the patterned hydrogels were measured on a NanoWizard II AFM (JPK Instruments, Berlin, Germany) in the force mapping mode with MLCT C tip cantilevers (Bruker, Palaiseau, France) with a nominal spring constant of 0.01 N/m. Young’s moduli were fitted from the indentation curves using a Hertz–Sneddon model [36 ]. Rigidity maps were sampled every 0.3 to 1 µm, depending on the size of the pattern.
The sizes of the rigid dots were determined via comparison to the AFM data. A new image was generated, composed of the sliding average of the AFM data on squares of 3 pixel sides. The limits of the stiff dot were set at places where the gradient of the sliding average was larger than 500 Pa/µm. This region was then filled with the largest disk. The center and the radius of this disk defined the geometric parameters of the stiff dots. The mean of the rigidity was calculated on this area. The average on the 3 to 5 dots was performed and taken as the rigidity of the stiff dots. The limit of the soft region was defined using the same methodology. The largest disk that fills the region limited by the edges of the soft background shares the same center as the disk that fits the stiff dot.

Atomic force microscopy images were collected using a JPK NanoWizard II AFM (JPK, Cambridge, UK) mounted on a Zeiss AxioObserver, in contact mode. Silicon nitride cantilevers (Veeco, Cambridge, UK) were used.

AFM has been used to observe at high resolution the effects of GO on bacteria morphology without altering the samples by complex dehydration and metallization. For AFM measurements, samples were prepared as explained elsewhere [36 (link)]. Briefly, 20 μL of GO were deposited on sterile cover glass slides, air-dried and washed once with ultrapure water (37 °C) to remove salt deposits interfering with the measurements. After sample preparation, measurements were immediately performed with a NanoWizard II AFM (JPK Instruments AG, Berlin, Germany). The images were acquired using silicon cantilevers with high aspect-ratio conical silicon tips (CSC37 Mikro-Masch, Tallinn, Estonia) characterized by an end radius of about 10 nm and a half conical angle of 20°. Cantilevers with a nominal spring constant of 0.4 n/m were accurately calibrated.