We used for the analysis of samples an ICON AFM from Bruker, with 30 pm noise, drift rates less than 200 pm per minute and sub-nanometer resolution in z. Ultra-sharp probes were used for ultra-high resolution with a radius of curvature at the tip as low as Rc = 5 nm, assuring an estimated maximum lateral resolution . Images were acquired in tapping mode in air setting a frequency of 0.5 Hz and a resolution of 512 × 512 pixels. Several measurements were performed for each sample setting scanning areas of 2 μm × 2 μm, 5 μm × 5 μm, 10 μm × 10 μm. Bi- and three- dimensional image elaboration was conducted using the version 1.40 of Nanoscope software (from Bruker). Force measurements and mechanical characterization of samples were performed using a scan size of 5 μm, with a ramp size of 670.0 nm and a ramp rate of 1 Hz, using an antimony n-doped silicon cantilever with spring constant of 42 N/m, (from TESPA).
Nanoscope software
Manufactured by Bruker
Sourced in United States, Germany
The NanoScope software is a core component of Bruker's atomic force microscopy (AFM) systems. It provides the fundamental functionality for controlling the AFM hardware, acquiring and processing data, and visualizing the resulting images.
Automatically generated - may contain errors
Lab products found in correlation
Nanoscope 5 controller, by Bruker (9 mentions)
Picoforce multimode afm, by Bruker (4 mentions)
Np s type d, by Bruker (3 mentions)
Originpro, by OriginLab (3 mentions)
Scanasyst air, by Bruker (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Tcs sp5 2, by Leica (2 mentions)
Mlct with a pyramidal tip, by Bruker (2 mentions)
Plan apochromat 63x 1.4 na oil immersion objective, by Zeiss (2 mentions)
Concanavalin a (cona), by Merck Group (2 mentions)
Effectene, by Qiagen (2 mentions)
Bl ac40ts probes, by Olympus (2 mentions)
Multimode 8, by Bruker (2 mentions)
Multimode 8 instrument, by Bruker (2 mentions)
Bioscope resolve, by Bruker (2 mentions)
Type e scanner head, by Bruker (2 mentions)
Sharpened tesp ss, by Bruker (2 mentions)
Bioscope catalyst afm, by Bruker (2 mentions)
Nanoscope analysis software, by Bruker (2 mentions)
Multimode 8 afm, by Bruker (2 mentions)
50 protocols using nanoscope software
1
Atomic Force Microscopy Characterization
2
Atomic Force Microscopy Data Processing
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All computations were performed using LabVIEW programming language (National instruments). Offline processing was partially done with the help of the SPIP software package (Image Metrology, Inc.). The unfiltered AFM data signals can be collected by using various methods described in Supplementary note 1 . The data can further be processed with a variety of external data processing software, for example Matlab, Mathcad or LabVIEW. It is possible to process the recorded signal in real time by using real-time data processing hardware. Some basic manipulations (adding and subtraction of images, multiplication by a constant) can be done off-line within the regular Bruker NanoScope software, or more advanced software processing (for example SPIP). It should be noted that the statement about faster work of ringing mode is applicable no matter if the collected data were processed off-line or online. The processing algorithms are described in the main text.
3
Atomic Force Microscopy Imaging of pDNA
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One milliliter of solutions of pDNA and the DMAPA-Glyp-2.8/pDNA complex (with weight ratios of 0.3, 0.5, 2, and 20) were prepared in the AFM buffer to a final concentration of 2.5 μg/mL for pDNA. The AFM buffer included Tris-EDTA buffer (10 mmol/L Tris-HCl and 1 mmol/L EDTA, pH 8.0) and 5 mmol/L MgCl2. To successfully image pDNA molecules and investigate the interactions between pDNA and the DMAPA-Glyp-2.8 derivative, pDNA molecules were extended following the methods used in a previous work.26 (link) The sample solution (20 μL) was dropped on a freshly cleaved mica surface and left to stand for 5 minutes. Then, the mica surface was tilted at 45° from level, allowing the solution to slide slowly to the bottom edge. The treated mica surface was washed with distilled water eight times by the same method. The distilled water was then evaporated at room temperature under vacuum for 2 weeks. AFM images of 256×256 pixels were performed on a MultiMode 8 atomic force microscope (Bruker Optics, Billerica, MA, USA) in tapping mode, and the images were analyzed using NanoScope software (Bruker). Silicon tips (TESP; Bruker) were used with a force constant of 40 N/m.
4
Cell Stiffness Measurement using AFM
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The stiffness of cells was measured using an atomic force microscope (Bruker NanoScope) coupled to a confocal microscope (TCS SP5II; Leica), as described previously50 (link),51 (link). The point-and-shoot procedure (NanoScope software; Bruker) was used to measure cell stiffness. All cells were kept in CO2-independent cell culture medium during the measurement. A fluorescent 10 μm polystyrene bead (Invitrogen) was glued to silicon nitride cantilevers with nominal spring constants of 0.06 N m−1 (NP-S type D; Bruker). Indentations were performed using the single force option with a total indentation depth of 50–100 nm. To obtain cell stiffness values from force curves, PUNIAS software was used as described previously50 (link),51 (link). Multiple force displacement curves (at five different locations) were fitted to the Hertz model to calculate cell cortical stiffness (Young’s modulus).
5
Studying Force-Induced Cytoskeletal Dynamics
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S2R+ cells were plated on glass coverslips coated with concanavalin A (Sigma, St Louis, MO) and transfected to express fluorescently tagged MyoII and βH-Spec using Effectene (Qiagen), per manufacturer’s instructions. Lateral indentation experiments were conducted 2 days after transfection with a modified Catalyst AFM integrated with an Axio Observer fluorescence microscope (Zeiss). To determine the effect of a localized mechanical force on MyoII and βH-Spec localization, the cantilever (MLCT with a pyramidal tip, Bruker) was first brought into full contact, at around 50 nN setpoint force, with the glass surface on a cell-free area within 10 μm from a target cell. Next, the cell was laterally translated into the stationary cantilever using the piezoelectric XY stage and the NanoScope software (Bruker). The cantilever tip indented the edge of the cell by 2–5 μm. Cells were simultaneously imaged with a plan-apochromat 63x/1.4 NA oil immersion objective (Zeiss). Time lapse images were taken at 5 second intervals using the Micro-Manager software (http://micro-manager.org/wiki/Micro-Manager ).
6
Atomic Force Microscopy of Thermosensitive Gels
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Atomic force microscopy images were obtained with a Catalyst BioScope (Bruker) coupled to a confocal microscope (TCS SP5II, Leica). Imaging was done in tapping mode using silicon nitride cantilevers with nominal spring constants of 0.06 N m–1 (S-NL type D, Bruker). The samples were prepared on a WillCo-dish glass bottom dish and heated to 37 °C using a LakeShore Model 331 temperature controller. Water was kept on top of the gel to prevent drying out. Each sample was kept at this temperature for at least 1 h prior to measurements at T > LCST. Root mean square (RMS) values were calculated using the roughness feature of NanoScope software (Bruker).
7
Gamma-Irradiated Plasmid DNA Visualization
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pBR322 plasmid was purchased from Fisher Scientific (Sweden) and irradiated at room temperature with gamma radiation in a stock solution that contained 10 mM TEN and 1 mM EDTA. Φ174 plasmid for ssDNA visualization was purchased form New England Biolabs (USA). The plasmid solution was heated in 40 mM HEPES buffer at 50°C for 3, 6 and 24 hours or kept at room temperature. The surface of mica was cleaved and pretreated with 10 mM NiCl2 in 40 mM HEPES for approximately 1 minute. Later, the mica was rinsed with MQ water, and 1 μg/ml DNA was applied at the respective temperature on the cleaved mica in 10 mM NiCl2 and 40 mM HEPES buffer. After 5 minutes, unbound DNA was washed away, and the mica surface was dried at room temperature or at 50°C. Next, the mica was stored in a closed box until analysis, when a buffer that contained 10 mM NiCl2 and 40 mM HEPES was added to the surface. Imaging was performed in liquid, using the peak force tapping mode. For imaging, ScanAsystFluid+ probe with 150 kHz resonance frequency and a spring constant of 0.7 N/m (Bruker, Massachusetts, USA) was mounted in a Dimension FastScan Bio system (Bruker, USA). Acquired images were analyzed using NanoScope software (v1.5, Bruker, USA).
8
Nanoindentation for Substrate Stiffness Measurement
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Substrate stiffness was measured by nanoindentation under an atomic force microscope (Bruker Nanoscope) using the “point and shoot” procedure (Nanoscope software, Bruker). A fluorescent polystyrene bead (φ = 10 μm, Invitrogen) was glued to silicon nitride cantilevers with nominal spring constants of 0.06 N/m (NP-S type D, Bruker). The system was calibrated in cell-free medium at 37 °C prior to each experiment by measuring the deflection sensitivity when pressing the cantilever onto a glass coverslip, which allowed the cantilever spring constant to be determined using the thermal noise method38 (link). For each gel, indentation force curves at 30 different locations on the gels were acquired. Before and during indentation experiments gels were kept in medium in 37 °C. To address local stiffness changes generated by cells on protein gels and PAAm gel, we applied spatially resolved AFM nanoindentation in live-cell culture by probing the matrix around single cells. To obtain stiffness values from force curves we used the PUNIAS software (http://punias.free.fr ). Specifically, we corrected for baseline tilt, and used the linear fitting option for the Hertz model with a Poisson ratio of 0.5 on the indentation curve.
9
Characterizing Patterned Protein Surfaces by AFM
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The AFM data
were collected on a Multimode 8 instrument equipped with a 15 μm
scanner (E-scanner) coupled to a NanoScope V controller (Bruker).
NanoScope software (v8.15, Bruker) was used for data collection, and
Gwyddion (v2.32, open source software covered by GNU general public
license,www.gwyddion.net ) and OriginPro (v8.5.1, OriginLab
Corp.) software packages were used for data processing and analysis.
The measurements of the patterned SAMs were performed in tapping mode
in air at ambient conditions by use of AC160TS probes (Olympus) with
a nominal spring constant of approximately 40 N·m–1 and a nominal resonant frequency of around 300 kHz.
The chemically
patterned surfaces with the immobilized protein molecules on them
were imaged in peak force tapping mode at nearly physiological conditions
in buffer (PBS, pH 7.4), at room temperature by use of BL-AC40TS probes
(Olympus). In this case, the Z-modulation amplitude
was adjusted to values in the range 20–24 nm, while the Z-modulation frequency was 2 kHz and the contact tip-sample
force was kept in the range 80–100 pN.
were collected on a Multimode 8 instrument equipped with a 15 μm
scanner (E-scanner) coupled to a NanoScope V controller (Bruker).
NanoScope software (v8.15, Bruker) was used for data collection, and
Gwyddion (v2.32, open source software covered by GNU general public
license,
Corp.) software packages were used for data processing and analysis.
The measurements of the patterned SAMs were performed in tapping mode
in air at ambient conditions by use of AC160TS probes (Olympus) with
a nominal spring constant of approximately 40 N·m–1 and a nominal resonant frequency of around 300 kHz.
The chemically
patterned surfaces with the immobilized protein molecules on them
were imaged in peak force tapping mode at nearly physiological conditions
in buffer (PBS, pH 7.4), at room temperature by use of BL-AC40TS probes
(Olympus). In this case, the Z-modulation amplitude
was adjusted to values in the range 20–24 nm, while the Z-modulation frequency was 2 kHz and the contact tip-sample
force was kept in the range 80–100 pN.
10
Atomic Force Microscopy of Contact Lenses
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Lenses were cut on four periphery edges to flatten the lens sample. Atomic force microscopy measurements were performed on a Dimension Icon Atomic Force Microscope (Bruker Nano, Inc., Billerica, MA) in contact mode, using Sharpe Nitride Lever probes SNL-10 (force constant 0.06 N/m). The scanned area of the imaging sample was 20×20 μm. Lens samples and the AFM tip were kept hydrated by standard lens packaging solution (0.9% sodium chloride with borate buffer, pH∼7.4). All measurements were obtained under controlled room temperature at relative humidity of 50%. For each CL, at least three areas of 20×20 μm of the pigmented regions, three areas of 20×20 μm of the central clear (within the optical zone) regions of either the front or the back surface were sampled, depending on the pigment location. We sampled at least three lenses across different powers (−1.00D, −3.00, and −6.00D) from each brand. The Bruker NanoScope software was used for image processing and interpretation. We used a flatten tool from the image analysis menu before obtaining roughness measurement. Atomic force microscopy surface roughness is described by two parameters: average RMS described the average surface roughness of a given area; and peak-to-peak RMS described the difference between the highest and the lowest points of RMS on the surface.
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