Atomic force microscopy (AFM) imaging of α‐syn fibrils was performed as previously described (Plotegher, Greggio, Bisaglia, & Bubacco, 2014 ) in a “PeakForce tapping” mode with Scanasyst‐Air probes (Bruker, Mannheim, Germany) on a Nanoscope V system equipped with a Multimode head and a type‐E piezoelectric scanner (Bruker, Mannheim, Germany). Ten microliters of sample were deposited on freshly cleaved mica (RubyRed Mica Sheets, Electron Microscopy Sciences, Fort Washington, USA) and left to adsorb for 5 min at room temperature (∼20°C). The mica surface was then rinsed with ∼500 μl of MilliQ H2O (Millipore Simplicity) at the same temperature and dried with dry nitrogen.
Nanoscope 5 system
Manufactured by Bruker
Sourced in Germany
The NanoScope V system is a high-performance atomic force microscope (AFM) designed for advanced research and development applications. It provides precise nanoscale imaging and characterization capabilities for a wide range of materials and samples.
Automatically generated - may contain errors
Lab products found in correlation
Milli q h2o, by Merck Group (1 mentions)
Rubyred mica sheets, by Electron Microscopy Sciences (1 mentions)
Scanasyst air probe, by Bruker (1 mentions)
Afm probe, by Bruker (1 mentions)
Nanoscope analysis 1, by Bruker (1 mentions)
Atomic force microscope, by Agilent Technologies (1 mentions)
Uv 2600, by Shimadzu (1 mentions)
6 protocols using nanoscope 5 system
1
Atomic Force Microscopy of α-Synuclein Fibrils
2
Visualizing Tau Protein Aggregation by AFM
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We used AFM to visualize the formation of molecular assemblies during the spontaneous aggregation of tau protein. Tau protein solutions were prepared and incubated as described for aggregation studies and diluted to 1 µM with 50 mM PB. 30 µl of each sample was spotted onto freshly cleaved muscovite mica disks (Assing) and incubated for 5 min. The mica disks were then washed with 5 ml Milli-Q water and dried under a gentle nitrogen flow. Measurements were performed using 0.01-0.025 Ohm/cm antimony-doped silicon probes (T: 3.5-4.5 µm, L: 115-135 µm, W: 30-40 µm, k: 20-80 N/m, f0: 323-380 kHz, Bruker AFM probes) on a Multimode AFM with a Nanoscope V system operating in tapping mode with a scan rate in the 0.5-1.2 Hz range, proportional to the area scanned. Measurements confirmed all topographic patterns on at least three separate areas. To exclude the interference of artefacts, freshly cleaved mica soaked in 30 µl 50 mM PB and containing 1 mM DTT and 11.5 µg/ml heparin were also analyzed as controls. To describe the aggregated structures, AFM sample images were analyzed for diameter and height with the Scanning Probe Image Processor (SPIP Version 5.1.6) data analysis package.
3
Fibril Disassembly Monitored by AFM
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Liquid cell and freshly cleaved mica were pre-cooled at 4 °C. Overall, 30 μl of fibril samples were added into the liquid cell. Images were acquired in fluid using a MultiMode AFM instruments with NanoScope V system (Bruker). Measurements were carried out using SNL-10 silicon cantilevers with a spring constant of 0.24 N m−1 (Bruker) in scanasyst mode. Disassembly of fibrils was monitored by AFM with a scan rate of 0.9 Hz upon temperature increase from 4 °C to room temperature. All images were analyzed by NanoScope Analysis 1.5 (Bruker).
4
Characterization of I-Fe3O4-NPs and I-Fe3O4-NBC
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The synthesized I-Fe3O4-NPs and I-Fe3O4-NBC were characterized using advanced analytical techniques, i.e., UV–visible, FT-IR, SEM, and XRD. Morphological characters of I-Fe3O4-NPs and I-Fe3O4-NBC were analyzed by scanning electron microscope (SEM) model JSM-6380 (JEOL Electronics Company, Japan). Similarly, the crystal structure was studied by X-ray diffraction (D-8 of Bruker). The spectrophotometric study for the confirmation of I-Fe3O4-NPs was conducted by a double-beam UV–visible spectrophotometer (UV-2600, Shimadzu, Japan). FT-IR spectra of I-Fe3O4-NPs nanoparticles and their composites in the range of 400–4000 cm−1 were obtained by FT-IR spectrophotometer Thermo Electron Scientific (Madison, WI, USA) with a KBr pellet. The size and shape of I-Fe3O4-NPs and I-Fe3O4-NBC was confirmed by atomic force microscope (Agilent, Santa Clara, CA, USA). AFM imaging was performed on the NanoScope V system (Bruker Ltd, Germany). Dynamic light scattering (DLS) and zeta potential measurements were taken for particle size and adsorption activity of adsorbent using the laser scattering particle size distribution analyzer (Horiba Scientific, Kyoto, Japan) and the zeta potential analyzer (ELSZ-2000), respectively. Furthermore, a salt addition method was used to determine the point of zero charges (isoelectric point) of the adsorbent, as reported elsewhere [53 (link)].
5
Atomic Force Microscopy Surface Analysis
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A NanoScope V system (Bruker, Karlsruhe, Germany) was used in contact mode at 1 Hz to assess parameters of surface roughness of each surface. The tips used (NT-MDT) had a 10 nm radius of curvature. The following parameters were analyzed with the Gwyddion v2.59 software: mean roughness (Sa), mean quadratic roughness (Sq), maximum pit depth (Sv), and maximum height (Sz).
6
AFM Imaging of Protein Monolayers
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AFM imaging was performed with NanoScopeV system (Bruker Inc.) in tapping mode in air, immediately after transfer of the protein monolayer from air/buffer interface to the glass surface. Standard silicon nitride (Si3N4) probe tips (Tap300Al-G, Budget Sensors, Innovative solutions Ltd., Bulgaria) were used (tip radius <10 nm). The hBest1-covered glass slides were fixed to the metal pads and scanned with rate 0.3 Hz. The images (512 × 512 pixels) were captured in height, deflection and phase mode and analyzed by NanoScope 6.13R1 software in the following order: first order flattening; determination of the length, width and height of the individual proteins; "deconvolution" of the lateral dimensions. The height of the objects was determined as the difference between the threshold value and the highest point of the protein. It is well established that the lateral dimensions of all imaged objects are broadened caused by the tip geometry which has a finite size close to the size of a protein molecule 5 ÷ 10 nm. This artefact is known as "tipdeconvolution effect" and has to be/can be removed by process called 'deconvolution' using the relation: D 4, where D is the real diameter of the protein molecule, R is the tip radius, and d is the diameter measured by AFM [28] .
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