Aβ incubated in the presence or absence of FapCS, FapC monomers, FapC fibrils or bLgS were subjected to TEM imaging. After incubation for 12 h, a drop of sample was applied on a glow‐discharged carbon‐coated copper grid. After 1 min, the sample was blotted and then negatively stained with uranyl acetate (1%) for 30 s. The dried grid was then imaged with a Technei F20 TEM at 200 kV. For AFM, a drop of sample was placed on freshly cleaved mica surface and rinsed with water after 2 min and dried with pressurized air. The sample was scanned with an AFM (Nanoscope VIII Multimode Scanning Force Microscopes, Bruker), covered with an acoustic hood to minimize the vibrational noise. Images were acquired under the tapping mode with a silicon nitride cantilever (Bruker). AFM images were processed with Nanoscope Analysis 1.5 software to flatten the background and to calculate the statistical parameters.
Silicon nitride cantilever
Manufactured by Bruker
Sourced in United States
Silicon nitride cantilevers are a type of lab equipment used in atomic force microscopy. They consist of a flexible silicon nitride beam with a sharp tip at the end, which is used to scan the surface of a sample. The cantilever's deflection as it interacts with the sample surface is measured, allowing for high-resolution imaging and surface characterization.
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Lab products found in correlation
Multimode 8 scanning probe microscope, by Bruker (2 mentions)
Nanoscope 8 multimode scanning force microscope, by Bruker (1 mentions)
Nanoscope analysis software, by Bruker (1 mentions)
Dimension icon, by Bruker (1 mentions)
Nanoscope analysis, by Bruker (1 mentions)
Bioscope catalyst afm system, by Bruker (1 mentions)
Eclipse te200, by Nikon (1 mentions)
Nanowizard 4 bioscience afm, by Bruker (1 mentions)
Mfp 3d bio, by Oxford Instruments (1 mentions)
Nanoscope 5 controller, by Bruker (1 mentions)
Scanasyst air, by Bruker (1 mentions)
Asylum mfp 3d afm, by Oxford Instruments (1 mentions)
9 protocols using silicon nitride cantilever
1
Visualizing Amyloid Fibril Formation
2
Surface Topology Scanning of Membranes
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The surface topology scans of the membranes were obtained using the atomic force microscope Bruker Dimension Icon (Bruker, Berlin, Germany) in tapping mode. Tapping mode was employed in order to obtain a high-resolution scans of surface details, using a Bruker SNL-10 type D (low stiffness) silicon nitride cantilever with a silicon tip of 2 nm tip radius. The cantilever’s dynamic properties, such as natural frequency and normal stiffness, were obtained using the thermal tune method where the thermal noise spectrum of the cantilever was measured and fitted to a Lorentzian harmonic oscillator model in the air, which corresponded to 22 kHz. The obtained calibration allows the usage of minimal contact force for the measurement, which has minimal impact on the surface, and thus the average used force was 1.5 pN. ±5%. The scans were made with scan sizes of 2 µm2 and 5 µm2 with 512 scan lines each, with 512 data points acquired per line. The obtained data were processed in order to obtain the values of surface roughness parameters after tilt and bow corrections using the proprietary Bruker Nanoscope Analysis software (Bruker, Berlin, Germany). The roughness is given as root-mean-square (RMS) roughness, defined as the standard deviation of the elevation within the given area.
3
Measuring Intracellular Stiffness via AFM
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AFM was used to measure intracellular stiffness as previously described [1, 2, 30] . Briefly, to measure intracellular stiffness, MEFs cultured on soft and stiff polyacrylamide hydrogels in phenol red-free DMEM with 10% FBS were indented with a silicon nitride cantilever (Bruker; spring constant, 0.06 N/m) with a conical AFM tip (40 nm in diameter). AFM in contact mode was applied to single adherent MEFs using a BioScope Catalyst AFM system (Bruker) mounted on a Nikon Eclipse TE 200 inverted microscope. To analyze the stiffness, the first 600 nm of horizontal tip deflection was fit with the Hertz model for a cone. 5 to 10 measurements of intracellular stiffness from each experiment were acquired near the periphery of each cell (8 to 10 cells per experimental condition). AFM curves were quantified and converted to Young's modulus (stiffness) using AFM analysis software (NanoScope Analysis, Bruker).
4
Tissue Stiffness Mapping of Mouse Inner Ear
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Fresh inner ear tissues from mice (E9.5, E13.5, E16.5, and E18.5) were embedded in OCT without fixation. Then, the samples were snap-frozen and sectioned into 40-μm-thick sections with a microtome. Sections were placed onto poly-lysine–coated slides and covered with PBS. Before AFM measurements, each sample was stained with Hoechst 33342 to label cell nuclei. Samples were mounted on an atomic force microscope system (JPK NanoWizard ULTRA Speed 2; Bruker) and subjected to tissue stiffness measurements. Silicon nitride cantilevers with a spring constant of 0.06 N/m (Bruker) were used, and a borosilicate glass spherical ball of 20 μm in diameter (Novascan Tech) was attached using epoxy glue (Araldite). Cantilevers were calibrated using the thermal oscillation method and were tapped on the stromal regions, which closely connect to epithelial regions. Tissue indentation was conducted with the following settings: piezo displacement speed 2 μm s−1 and sampling rate 2000 Hz with a maximum force of 300 pN. Ten 30 μm by 30 μm AFM force maps were typically obtained at each stage, and each stiffness map was presented as a 5 × 5 raster series of indentations using the JPK software. Samples were assumed to be incompressible, a Poisson’s ratio of 0.5 was used, and Young’s modulus of each sample was calculated using a fit of the Hertz model.
5
Mechanical Properties of Klebsiella pneumoniae under CSA Treatments
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Characterization of mechanical properties of Klebsiella pneumoniae BAA-2473 cells untreated and treated with 1 μg/mL, 5 μg/mL, and 10 μg/mL of CSA-13, CSA-44, and CSA-131 were performed using an atomic force microscope NanoWizard 4 BioScience AFM (JPK Instruments, Bruker) equipped with a liquid cell setup. Silicon Nitride cantilevers (Bruker MSCT) described by a spring constant of 0.37 N/m were used. Due to the lateral forces during contact mode scanning, the force curves-based imaging mode with the resolution of 128 pixels per line was used, to image bacterial surfaces (JPK QI™ mode - Quantitative Imaging). The topography maps sizes of 5 μm×5 μm and 3 μm×3 μm were recorded. To assess the wider spectrum of cells changes after treatment, QI maps were used to determine bacteria surface stiffness (a slope mode) and adhesion forces between the cells and the AFM probe.
6
Tapping Mode AFM Imaging of Biomolecules
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AFM experiments
were carried out on a Multimode 8 Scanning Probe Microscope (Bruker,
U.S.A.) covered with an acoustic hood to minimize vibrational noise.
A droplet of the different aliquots was deposited onto freshly cleaved
mica, incubated for 2 min, rinsed with Milli-Q water, and dried under
nitrogen. The AFM was operated in tapping mode under ambient conditions
using commercial silicon nitride cantilevers (Bruker, U.S.A.) at a
vibration frequency of 150 kHz. Images were simply flattened using
Nanoscope 8.1 software, and no further image processing was carried
out. The resulting images were used for the statistical analysis.
AFM images were traced using FiberApp software.22 (link)
were carried out on a Multimode 8 Scanning Probe Microscope (Bruker,
U.S.A.) covered with an acoustic hood to minimize vibrational noise.
A droplet of the different aliquots was deposited onto freshly cleaved
mica, incubated for 2 min, rinsed with Milli-Q water, and dried under
nitrogen. The AFM was operated in tapping mode under ambient conditions
using commercial silicon nitride cantilevers (Bruker, U.S.A.) at a
vibration frequency of 150 kHz. Images were simply flattened using
Nanoscope 8.1 software, and no further image processing was carried
out. The resulting images were used for the statistical analysis.
AFM images were traced using FiberApp software.22 (link)
7
Measuring Hydrogel Stiffness via AFM
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Atomic force microscopy (AFM, MFP-3D-Bio; Asylum Research, Santa Barbara, CA, USA) was applied to determine the stiffness of GelMA hydrogels. The hydrogel samples were incubated in PBS at 37 °C for 24 h and then their stiffness was measured at room temperature. An optical microscope was used to control the position of the AFM tip. Silicon nitride cantilevers (Bruker, Camarillo, CA, USA) with a 600 nm diameter glass ball were used as the probe. The exact spring constant was measured before each experiment using a thermal tuning method. The force curves were collected and fitted to Hertz’s contact model to calculate the Young’s modulus [8 (link)]. Nine samples of each group were measured for calculation of means and standard deviations.
8
Nanoscale Visualization of Provox Vega Prosthesis
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Segments were taken from the valve and flange of a Provox Vega voice prosthesis and mounted onto 15 mm AFM specimen disks (Agar Scientific, F7003) with superglue. These surfaces were imaged at ambient temperature using a Bruker Multimode 8 scanning probe microscope with a Nanoscope V controller, using the ScanAsyst peak-force tapping mode with a 50 μm × 50 μm scan area and a 700 nm scan height. SCANASYST-AIR (Bruker) silicon nitride cantilevers (tip height of 2.5–8.0 μm, nominal tip radius of 2 nm) with a nominal spring constant of 0.4 N/m and a resonance frequency of 70 kHz were used throughout. The image acquisition software was Nanoscope 8.15 R3sr5 and the processing software was Nanoscope Analysis.
9
Single-Molecule Force Spectroscopy of Titin I27
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Single-molecule force spectroscopy experiments were performed using an Asylum MFP-3D AFM (Asylum Research, Santa Barbara, CA), using silicon nitride cantilevers with spring constants in the range 40.28 ± 1.28 pN nm -1 (Bruker, CA). Cantilever spring constants were calibrated in buffer using the equipartition theorem [56, (link)57] (link).
The protein used was a double mutant of the I27 domain from human cardiac titin, where both cysteine residues were mutated to serines, which will be referred to as I27 in this paper. (I27) 5 polyprotein constructs were expressed and purified according to methods described previously [58] (link). All domains in the protein constructs were assumed to be folded under the conditions used in the AFM experiments, based on characterization of the stability of (I27) 5 described elsewhere [58] (link).
A total of 30-50 μl of 0.1 mg ml -1 protein solution in sodium phosphate buffer (63 mM, pH 7.4) was applied onto a coverslip with a freshly stripped gold surface, and the polyproteins immobilized on the surface by covalent attachment of the sulphydryl groups of two cysteine residues at the C-terminus of the polyprotein chain. After 15 min, the surface was flushed with fresh buffer to remove any unbound protein. Results are shown from mechanical unfolding experiments collected in triplicate at pulling speeds of 160, 400, 1000, and 2000 nm s -1 .
The protein used was a double mutant of the I27 domain from human cardiac titin, where both cysteine residues were mutated to serines, which will be referred to as I27 in this paper. (I27) 5 polyprotein constructs were expressed and purified according to methods described previously [58] (link). All domains in the protein constructs were assumed to be folded under the conditions used in the AFM experiments, based on characterization of the stability of (I27) 5 described elsewhere [58] (link).
A total of 30-50 μl of 0.1 mg ml -1 protein solution in sodium phosphate buffer (63 mM, pH 7.4) was applied onto a coverslip with a freshly stripped gold surface, and the polyproteins immobilized on the surface by covalent attachment of the sulphydryl groups of two cysteine residues at the C-terminus of the polyprotein chain. After 15 min, the surface was flushed with fresh buffer to remove any unbound protein. Results are shown from mechanical unfolding experiments collected in triplicate at pulling speeds of 160, 400, 1000, and 2000 nm s -1 .
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