Purified tetranucleosome and dodecamer arrays were diluted to 10 nM and crosslinked with 1% formaldehyde for 1 h at room temperature. Crosslinked sample was dialyzed against 20 mM HEPES-NaOH pH 7.5; 1 mM EDTA; 1 mM DTT, and centrifuged at 20,000 x g to remove aggregates. Crosslinked nucleosome array samples were diluted to 1 nM using 10 mM MOPS pH 7.0 and 5 mM MgCl2, and 3 μL of sample were deposited and incubated for 2 min on freshly cleaved bare mica V1 (Ted Pella Inc.), after which was rinsed with Milli-Q water, and then gently dried under a stream of N2 perpendicular to the mica surface. AFM micrographs were taken with a MultiMode NanoScope 8 atomic force microscope (Bruker Co.) equipped with a vertical engagement scanner E. The samples were excited at their resonance frequency (280-350 kHz) with free amplitudes (Ao) of 2-10 nm and imaged in tapping mode using silicon cantilevers (Nanosensors). The image amplitude (set point As) and A0 ratio (As/A0) was kept at ~0.8 in a repulsive tip-sample interaction regime, and phase oscillations were no greater than ± 5 degrees. The surface was rastered following the fast scan axis (x) at rates of 2 Hz, capturing the retrace line to reconstruct the AFM micrographs. All samples were scanned at room temperature in air, at a relative humidity of 30%.
Silicon cantilevers
Manufactured by Nanosensors
Sourced in United States
Silicon cantilevers are microscale mechanical structures fabricated from silicon. They are designed to function as sensitive force-sensing elements in various analytical and measurement applications.
Automatically generated - may contain errors
Lab products found in correlation
Bare mica v1, by Ted Pella (2 mentions)
Multimode 8 nanoscope atomic force microscope, by Bruker (2 mentions)
Pico plus 5500 ilm afm, by Agilent Technologies (1 mentions)
Pico view1.4 version software, by Agilent Technologies (1 mentions)
Pico image advanced version software, by Agilent Technologies (1 mentions)
5 protocols using silicon cantilevers
1
Imaging Crosslinked Nucleosome Arrays
2
Atomic Force Microscopy of Liposomes
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For atomic forced microscopy (AFM) imaging of liposomal samples, 10 µl of the samples were deposited onto freshly cleaved muscovite Ruby mica sheets (ASTM V1 Grade Ruby Mica from MICAFAB) for 15–20 minutes. Mica sheets are basically negatively charged so samples bind strongly on the mica surface. After 15 min, the samples were dried by using a vacuum dryer. Sometimes the samples were gently washed with 0.5 ml Milli-Q water to remove molecules that were not firmly attached to the mica and the samples were dried as mentioned above. Acoustic alternative current mode AFM was performed using a Pico plus 5500 ILM AFM (Agilent Technologies, USA) with a piezoscanner maximum range of 9 µm. Micro fabricated silicon cantilevers of 225 µm in length with a nominal spring force constant of 21–98 N/m were used from Nano sensors, USA. Cantilever oscillation frequency at 150–300 kHz was tuned into resonance frequency. The images (512 by 512 pixels) were captured with a scan size between 0.5 and 2 µm at a scan speed rate of 0.5lines/S. Images were flattened using Pico view1.4 version software (Agilent Technologies). Image processing and analyzation was done through Pico Image Advanced version software (Agilent Technologies).
3
Atomic Force Microscopy of Photoinhibited Grana
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Four different types of samples were imaged: WT low-light-acclimated (WT) and photoinhibited (WT-PI) grana membranes; and soq-1 mutant low-light-acclimated (soq1) and photoinhibited (soq1-PI) membranes. Grana aliquots were deposited on freshly cleaved mica (10 mM tris-HCl pH 7.5, 150 mM KCl and 25 mM MgCl2), and incubated at room temperature for 1–3 hours. Mica was rinsed with water ten times and dried under N2 gas flow. AFM measurements were performed with a Multimode AFM Nanoscope V (Bruker Co.). The samples were imaged in tapping mode; the silicon cantilevers (Nanosensors) were excited at their resonance frequency (280–350 kHz) with free amplitudes of 2–15 nm. The image amplitude (set point As) and free amplitude (A0) ratio (As/A0) was kept at ∼0.8. All samples were imaged at room temperature in air, at a relative humidity of 30%. More than eight different grana patches were scanned per each type of membrane. Bi-layered patches were fully mapped at scans of 500 nm×500 nm. Higher resolution of 150 nm×150 nm images were also recorded.
4
Atomic Force Microscopy of Crosslinked Tetranucleosomes
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Purified tetranucleosomes were diluted to 10 nM and crosslinked with 1% formaldehyde for 1 h at room temperature. Crosslinked sample was dialyzed against 20 mM HEPES-NaOH pH 7.5; 1 mM EDTA; 1 mM DTT, and centrifuged at 20,000 x g to remove aggregates. Crosslinked tetranucleosome sample was diluted to 1 nM using 10 mM MOPS pH 7.0 and 5 mM MgCl2, and 3 µL of sample were deposited and incubated for 2 min on freshly cleaved bare mica V1 (Ted Pella Inc.), after which was rinsed with Milli-Q water, and then gently dried under a stream of N2 perpendicular to the mica surface. AFM micrographs were taken with a MultiMode NanoScope 8 atomic force microscope (Bruker Co.) equipped with a vertical engagement scanner E. The samples were excited at their resonance frequency (280-350 kHz) with free amplitudes (Ao) of 2-10 nm and imaged in tapping mode using silicon cantilevers (Nanosensors). The image amplitude (set point As) and A0 ratio (As/A0) was kept at ~0.8 in a repulsive tip-sample interaction regime, and phase oscillations were no greater than ± 5 degrees. The surface was rastered following the fast scan axis (x) at rates of 2 Hz, capturing the retrace line to reconstruct the AFM micrographs. All samples were scanned at room temperature in air, at a relative humidity of 30%.
5
Probing Molecular Interactions on Calcite
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Experiments were performed under ultrahigh vacuum (UHV) with a base pressure of 10 -10 mbar. Calcite crystals (purchased at Korth Kristalle) were prepared by first annealing at 250 °C for 3 h, then in situ cleaving, and finally annealing at 200 °C for 45 to 90 min. Molecules were deposited for 3 to 6 min from a Knudsen cell at a temperature of about 90 °C, while keeping the sample at room temperature. AFM measurements were performed at room temperature with a VT-AFM (ScientaOmicron), operated in the frequency modulation mode. We used silicon cantilevers from Nanosensors with eigenfrequencies of about 300 kHz in UHV and spring constants on the order of 40 N/m. Illumination of the sample was done with (1) a UV/vis lamp emitting between 300 and 600 nm and a power density of 3.09 mW/ cm 2 through a (Silux) UHV window with >90% transmission and (2) a 450 nm laser (spot size: about 19 mm 2 with a total power of 4.5 mW) through a borosilicate window with maximum transmission (about 90%) between 400 nm and 2 μm (see Supporting Information).
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