Cell stretcher and chamber holders were based on the design described in Faust et al. [22 (link)]. Major alterations due to space limitations below the AFM head (Nanowizard 1, JPK, Berlin, Germany) are described in the results section. Chambers were stretched by 7.5% of their size (corresponding to 1.5 mm) before experiments to avoid sagging of the chamber bottom. Strain was increased at a constant speed of 0.75%/s (0.15 mm/s) until the desired target strain was reached, and the system was arrested. Target strains were 25% and 50%, and the measurement period at each strain was 30 min. Due to the design of the chamber and the stretcher, the strain was uniaxial.
Nanowizard 1
Manufactured by Bruker
Sourced in Germany
The Nanowizard I is an atomic force microscope (AFM) system designed for high-resolution topographic imaging and nanoscale measurements. It is capable of operating in a variety of imaging modes to accommodate different sample types and research applications.
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Lab products found in correlation
Nanoscope 5 multimode afm, by Veeco (1 mentions)
Dnp 10, by Bruker (1 mentions)
Quanta 250 feg, by Thermo Fisher Scientific (1 mentions)
Lext ols3000, by Olympus (1 mentions)
Polystyrene microspheres, by Thermo Fisher Scientific (1 mentions)
Matlab, by MathWorks (1 mentions)
Jem 2100f, by JEOL (1 mentions)
Jsm 7100f, by JEOL (1 mentions)
12 protocols using nanowizard 1
1
Uniaxial Cell Stretching Methodology
2
Cell Compliance Measurement via AFM
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Measurements of cell compliance were conducted on a Nanowizard-1 (JPK Instruments, Berlin, Germany) atomic force microscope operating in force spectroscopy mode mounted on an inverted optical microscope (IX-81; Olympus, Tokyo, Japan). Atomic force microscopy (AFM) pyramidal cantilevers (MLCT; Bruker, Camarillo, CA, United States) with a spring constant of 0.03 N/m (nominal stiffness reported by the manufacturer) were used with a 15-μm diameter polystyrene bead attached at room temperature. Before conducting measurements, cantilever sensitivity was calculated by measuring the force–distance slope in the AFM software on an empty petri dish region. Cells were seeded on fibronectin-coated glass fluorodishes and allowed to spread for >2 h. Cell attachment to the substrate was confirmed by visual inspection before conducting the nanoindentation procedure. For each cell analyzed, force curves were acquired at an approach speed of 5 μm/s and a maximum set point of 1 nN. Force curves were taken in regions distal from the cell nucleus to avoid assessing nuclear stiffness. The force–distance curves were used to calculate elastic moduli in the AFM software through the application of the Hertz contact model (Harris and Charras, 2011 (link)).
3
Atomic Force Microscopy of Protein Samples
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The protein solutions were cast on freshly cleaved mica surfaces at room temperature and allowed to dry for 24 h. AFM was performed in tapping mode using a Nanowizard I atomic force microscope (JPK Instruments, Berlin, Germany). The scan rate was maintained at 1 Hz. Images were collected with a scanning window of 2 μm and analyzed using Nanoscope Analysis 1.5 software.
4
Topographical Analysis of PCL Films
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For the topographical analysis of PCL-derivative micrometric films, an AFM instrument Nanowizard I (JPK Instruments, Germany) was operated in air, using contact imaging mode at constant loading forces and rates. Cantilevers were calibrated before each experiment by means of thermal tune method. Silicon-nitride probes (DNP-10, Bruker, USA) with a nominal spring constant of 0.12 N m−1 were used in the experiments. All images were processed by JPK data analysis software.
In order to extract topographical information of S-layer coated samples, a Nanoscope V multimode AFM (Veeco, Santa Barbara, USA) was employed. Before use, the fluid cell was washed overnight with 2% SDS, rinsed thoroughly with ultrapure water, and dried with nitrogen. Topography images were recorded in tapping mode, at 1 Hz in 100 mM NaCl aqueous solution and at room temperature. The final setpoint was carefully controlled to avoid coating damaging. Silicon nitride (Si3N4) cantilevers of 0.32 N m−1 with sharpened tips (DNPS, Veeco) and gold-coated reflective back sides were cleaned in ethanol before use.
In order to extract topographical information of S-layer coated samples, a Nanoscope V multimode AFM (Veeco, Santa Barbara, USA) was employed. Before use, the fluid cell was washed overnight with 2% SDS, rinsed thoroughly with ultrapure water, and dried with nitrogen. Topography images were recorded in tapping mode, at 1 Hz in 100 mM NaCl aqueous solution and at room temperature. The final setpoint was carefully controlled to avoid coating damaging. Silicon nitride (Si3N4) cantilevers of 0.32 N m−1 with sharpened tips (DNPS, Veeco) and gold-coated reflective back sides were cleaned in ethanol before use.
5
Nanoparticle Effects on Gastric Cancer Stem Cells
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To evaluate the inhibitory effects of the various nanoparticles on gastric CSCs, CD44+ cells seeded in 96-well plates were exposed to a series of concentrations of the various nanoparticles in OPTI-MEM for 48 h at 37°C. Their antiproliferative activity was measured using CCK-8 assays, according to the manufacturer’s protocols. The morphological changes and ultrastructure of CD44+ cells treated with the various nanoparticles were investigated by atomic force microscopy (AFM, JPK NanoWizardI, Germany).
6
Measuring Cell Elasticity via AFM Indentation
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For the AFM indentation experiments a Nanowizard I (JPK Instruments, Berlin) equipped with a CellHesion module was used. Arrow-T1 cantilevers (Nanoworld, Neuchatel, Switzerland) were modified with polystyrene beads (radius 2.5 μm, Microparticles GmbH, Berlin, Germany) with the aid of epoxy glue to obtain a well-defined spherical indenter geometry and decrease local strain during indentation. The cantilever was lowered with a speed of 5 μm s−1 until a relative set point of 2.5 nN was reached. The resulting force distance curves were analysed using JPK image processing software (JPK instruments). Force distance data were corrected for the tip sample separation and fitted with a Hertz model for a spherical indenter to extract the apparent Young's Modulus68 (link). A Poisson ratio of 0.5 was assumed. A 2-h time window was required for measurement of each experimental condition.
7
Comparative TEM Sample Preparation Analysis
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To compare the sections prepared by ultramicrotomy and the TEM lamellas prepared by in situ FIB lift-out, as well as to demonstrate specific preparation artefacts, a further microscopic examination was performed. The data used to evaluate TEM sample preparation were obtained by scanning electron microscopy (Quanta FEG250; FEI, Frankfurt am Main, Germany) at a primary beam energy ranging from 5 to 10 keV with a large field detector (LFD). Surface morphology was examined using reflected-light microscopy (LEXT OLS 3000; Olympus, Hamburg, Germany), confocal laser scanning microscopy (LEXT OLS 3000; Olympus, Hamburg, Germany) and atomic force microscopy (Nanowizard I®, JPK-Instruments, Berlin, Germany). High-resolution AFM images were generated in contact mode using a standard CSC37 cantilever (Ultrasharp CSC37/ no AL, MicroMasch, Tallin, Estonia) with the following parameter settings: cut-off frequency, 150 Hz; amplification factor, 0.05; scan size from 100 µm × 100 µm to 2 µm × 2 µm; scan rate, 0.5 Hz; setpoint, 0.5 V at Vsum = 1.5 V; resolution, 512 × 512. AFM measurements were performed using JPK data-processing software (v.5.0.97).
8
Atomic Force Spectroscopy of hMSCs
9
Measuring Fibroblast Elastic Modulus
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To check the elastic modulus, the fibroblasts were embedded in collagen type I. After 3 days, the edge of gels was cut with scalpel and gently moved to cover glass. The elastic modulus of the gels was measured by using Atomic force microscopy (AFM) in Yonsei Center for Research Facilities. AFM measurements were performed with a JPK Nanowizard-I (JPK instruments) interfaced to an inverted optical microscope. Pyramidal C-AFM cantilever was used with 10 μm tip sphere. Before the experiments, the sensitivity of the cantilever was set by measuring the slope of force-distance curves on empty region of glass. Using the optical microscope, the tip of the cantilever was aligned over three regions in each gel. The data represented on the average of Young’s modulus.
10
Hydrogel Mechanical Characterization via AFM
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Force spectroscopy experiments were conducted at the atomic force microscope (AFM) Nanowizard® I (JPK Instruments, Berlin, Germany) in a custom-built liquid cell (diameter 2 cm, height 0.5 cm). Thin slices (1–2 mm) of swollen hydrogels were cut from the bulky samples with a scalpel and immobilized at the bottom of the cell by using two component epoxy glue (UHU Endfest 300, UHU GmbH & Co. KG, Bühl, Germany). All measurements were performed in Milli-Q-water at room temperature. As a probe a tipless silicon nitride cantilever (NSC 12, no Al coating, MikroMasch, Tallinn, Estonia) was used with a glass sphere (35 µm in diameter, Polysciences Europe GmbH, Eppelheim, Germany) attached to its front (colloidal probe). Before the actual measurements, the cantilevers were calibrated against the non-deformable glass substrate to determine their optical lever sensitivity resulting as the slope of the recorded force–displacement curve. The deformation of the sample was obtained by subtraction of the bending of the cantilever from the raw displacement data. The spring constant of the cantilever (0.56 N/m) was deduced from its thermal noise spectrum prior to the attachment of the colloidal probe [66 (link)].
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