We follow the previously published protocol to prepare samples for AFM imaging (72 (link), 73 , 74 (link), 75 (link)). Briefly, reconstituted nucleosomes were incubated in 200 mM NaCl and 10 mM Tris–HCl, pH 7.6, for 1 min on ice and then deposited on poly-l -lysine (0.01% w/v)–coated muscovite mica for 30 s, followed by 20 ml Milli-Q water rinsing, and drying with a gentle stream of filtered nitrogen gas. AFM imaging was performed on a Nanowizard Ultraspeed 2 (JPK) with AFM cantilevers, FASTSCAN-A (resonance frequency 1400 kHz, spring constant 18 N/m; Bruker) for high-speed imaging in air. All AFM images were acquired in tapping mode at room temperature. The scans were recorded at 3 Hz line frequency over a field of view of 3 μm × 3 μm at 2048 × 2048 pixels. For image processing, Scanning Probe Image Processor (SPIP, version 6.4; Image Metrology) was employed. Image processing involved background correction by using global fitting with a third-order polynomial and line-by-line correction through the histogram alignment routine.
Nanowizard ultraspeed 2
Manufactured by Bruker
Sourced in Germany
The Nanowizard Ultraspeed 2 is an atomic force microscopy (AFM) system designed for high-speed imaging and nanoscale characterization. It features a high-speed scanner and advanced control electronics for rapid data acquisition and processing.
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4 protocols using nanowizard ultraspeed 2
1
Nucleosome Preparation for High-Speed AFM Imaging
2
Nanoscale Surface Characterization
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All the samples were stuck to a glass slide with fast-setting cyanoacrylate glue. Each glass slide was viewed under an AFM (JPK NanoWizard®ULTRA Speed 2) using contact mode imaging. Then, the data were analyzed statistically using one-way ANOVA and post hoc tests [Figures 1 -4 ].
3
Analyzing Antimicrobial Effects on Gutta-Percha
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GP cones were randomly selected from the same batch (ISO size 30) for the study before expiration date. All the samples were sectioned 3 mm from their tip and stick to a glass slide with fast setting cyanoacrylate glue. Following this procedure, the samples were divided into one control and three treatment groups as following:
Group I – 10 untreated GP points (control group)
Group II – 10 GP points immersed in 5.25% NaOCl (Modi Surgico Healthcare Pvt. Ltd, Maharashtra, India) for 1 min
Group III – 10 GP points immersed in 70 μg/ml (0.007%) AgNPs (Nano Wings Pvt. Ltd, Khammam, Telangana, India) for 1 min
Group IV – 10 GP points immersed in 1.5 mg/ml ChNPs (AURA Biotechnologies Pvt. Ltd, Chennai, India) for 1 min.
4
Visualizing Plasmid DNA Topology by AFM
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AFM imaging of plasmid DNA was performed as described previously (67–69 (link)). In brief, for the AFM imaging, we deposited 20 μl of DNA at different topological states in TE buffer at a final concentration of 1 ng/μl on freshly cleaved poly-l -lysine (Sigma Aldrich, diluted to 0.01% in milliQ water; PLL)-coated muscovite mica. The sample was incubated 30 s before washing with 20 ml MilliQ water and drying with a gentle stream of filtered argon gas. After drying, the AFM images were recorded in tapping mode at room temperature using a Nanowizard Ultraspeed 2 (JPK, Berlin, Germany) AFM with silicon tips (FASTSCAN-A, drive frequency 1400 kHz, tip radius 5 nm, Bruker, Billerica, Massachusetts, USA). Images were scanned over different fields of view with a scanning speed of 5 Hz. The free amplitude was set to 10 nm. The amplitude setpoint was set to 80% of the free amplitude and adjusted to maintain a good image resolution. AFM image post-processing was performed in the software SPIP (v.6.4, Image Metrology, Hørsholm, Denmark) to flatten and line-wise correct the images (Supplementary Figure S2 ).
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