Custom made fibronectin-coated micro-patterned 1D lines and 2D areas (cat #1D2D EDDY) were purchased from CYTOO Inc. In addition, CYTOO chips Motility (CYTOO Inc., 38,040 Grenoble, France) was used as described [28 (link)]. Dimensions of 1D lines were evaluated with AFM. All AFM measurements were taken with the Bruker Bioscope Catalyst. To understand the nanoscale topography of these lines, the chips were measured using ScanAsyst probes in both contact mode and PF-QNM. Different methods were used to ensure that the topography was not an artifact of the large differences in the adhesive properties of the lines. These measurements were performed in air, PBS, and 10× PBS, which would reduce any charge interactions between the probe and the substrate. In every condition, the scans of the surface revealed that the fibronectin lines were a mean thickness of 2.5 µm and a depth of 3.5 nm (Figure S1 ).
Bioscope catalyst
Manufactured by Bruker
Sourced in United States, Germany
The Bioscope Catalyst is a high-resolution atomic force microscope (AFM) designed for bioscience applications. It provides nanoscale topographical and mechanical characterization of biological samples, including cells, proteins, and biomaterials. The Bioscope Catalyst is capable of operating in a variety of imaging modes to accommodate diverse research needs.
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Lab products found in correlation
Axio observer z1, by Zeiss (7 mentions)
Nanoscope analysis 1, by Bruker (6 mentions)
Nanoscope analysis software, by Bruker (5 mentions)
Scanasyst air cantilever, by Bruker (5 mentions)
Observer z1, by Zeiss (4 mentions)
Mlct d, by Bruker (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Nsc36 tip b, by MikroMasch (3 mentions)
Nanoscope analysis, by Bruker (3 mentions)
Tcs sp5 2, by Leica camera (3 mentions)
Vt1000, by Leica camera (2 mentions)
Arrow tl2, by NanoWorld (2 mentions)
Snl d cantilevers, by Bruker (2 mentions)
Agarose solution, by Meridian Bioscience (2 mentions)
Nanoscope software, by Bruker (2 mentions)
Nanoscope analysis v1, by Bruker (1 mentions)
Mlct silicon nitride cantilevers, by Bruker (1 mentions)
1 dodecanethiol, by Merck Group (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Tm4000plus, by Hitachi (1 mentions)
81 protocols using bioscope catalyst
1
Characterization of Fibronectin Micro-Patterned Surfaces
2
Nanomechanical Mapping of Nanofiber Scaffolds
3
Quantifying Surface Porosity via Atomic Force Microscopy
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The experiments were conducted by means of an advanced scanning probe microscope (Bioscope Catalyst, Bruker Inc., Santa Barbara, CA, USA), implemented on an inverted optical microscope (Zeiss Observer Z1, Zeiss, Jena, Germany). The whole system is placed on an insulating base to minimize effects of environmental mechanical vibrations on measurements. The experiments were performed using a V-shaped Bruker’s Sharp Microlever (MSNL, Bruker Inc., USA), that consists in high sensitivity Silicon Nitride cantilever. The topographic images were acquired on 50 × 50 µm scan area at resolution of 512*512. The images were analyzed by NanoScope Analysis software (Bruker Inc., USA) in order to quantify roughness parameters, and thus evaluate porosity at surface level. The roughness was expressed as Rq. In details, Rq was calculated as root mean square of height fluctuations respect to mean height value obtained by all data image, previously treated with a second order plane fit and with a second order flattening for deleting every bow and minimizing tridimensionality effects. To obtain a more accurate estimate of local roughness, Rq value was reckoned as the mean value of 20 selected areas of 3 × 3 um.
4
AFM Analysis of Crosslinked Biomaterial Coatings
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AFM analysis of the substrates was conducted using a Bruker BioScope Catalyst (Bruker Nano Surfaces, Santa Barbara, CA, USA) coupled with an inverted optical microscope (Axio Observer Z1 from Zeiss, Oberkochen, Germany). A low thermal energy probe was used (FastSscan-Dx, Bruker Probes) with a nominal tip radius of 8 nm and with a 0.25 N/m spring constant. The resonant frequency of the probe in liquid was approximately 110 kHz. Images were obtained in tapping mode at a typical line frequency of 1 Hz. Measurements were performed in sterile water at room temperature only for the following selected 12-layer systems: 1–(DR/CS)6 non-photocrosslinked; 2–(DR/CS)6 photocrosslinked; 3–(DR/CS)6 + rhBMP-2E; 4–(DR/CS)6 + rhBMP-2C. The detailed description of AFM analysis can be found elsewhere [57 (link)].
5
Topographic Analysis of Vγ9Vδ2 T Cells
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The topographic properties of Vγ9Vδ2 T cells after chitosan and CSNPs (0, 50 μg/mL) treatments were detected using AFM (Atomic Force Microscope). The methodology for AFM imaging has been described in detail in our previous publications.13 (link)–15 (link) In our measurements, tapping mode AFM (Bioscope Catalyst, Bruker) was used to image cells at room temperature. The spring constant of the cantilever was calibrated at ~0.6 N/m. Obtained images were analyzed using instrument equipped analysis software.
6
Atomic Force Microscopy Analysis of Cell Mechanics
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Cells were incubated with GO for 3 hours and then fixed in 3.7% paraformaldehyde in a 6-cm plastic dish. The cells with or without GO treatment were scanned by using an AFM (Bruker BioScope Catalyst) at a frequency of 1 Hz. To avoid confusing results regarding the heterogenicity on roughness and rigidity, we primarily scanned the whole cell and excluded the nucleus region via the AFM height analysis. Subsequently, the near-edge region (2.7 μm by 2.7 μm) as far from the nucleus as possible was scanned and recorded. For the roughness measurement, a plane fit was applied, and then, the statistical values (Rq) were calculated according to the heights of each pixel in the image by using NanoScope Analysis software. In addition, the 3D image was also provided with color-coded height information. To compute the Young’s modulus of cell surface, the force curves were measured, and an indentation model (Sneddon conical) was fitted to the force curves.
7
Nanomechanical Analysis of Nasoseptal Cartilage
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Dissected nasoseptal cartilage explants, previously stored at −80 °C, were analysed in PBS buffer at 37 °C using a Bioscope Catalyst (Bruker Instruments, Santa Barbara, California, USA). Bruker MLCT silicon nitride cantilevers (20 nm tip radius) were calibrated on clean glass slides and used for force spectroscopy and sample imaging in PeakForce modality. For each sample, at least 5 different areas were analysed, where each area ranged from 25 to 400 μm2. Image offline processing included low-order flattening and plane fitting using Nanoscope Analysis, v.1.50. Fibril diameter was measured using ImageJ on AFM topographical data with a total of 15 fibrils per sample.
8
Yeast Cell Adhesion Measurement
9
Atomic Force Microscopy of Alpha-Synuclein Fibrils
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A 10 μL volume of the
five times diluted aggregated sample (initial αS concentration
= 50 μM and N-protein concentration = 0.2–1 μM)
was deposited onto freshly cleaved mica (Muscovite mica, V-1quality,
EMS, US) and left to rest for 5 min. Then, the sample was carefully
washed four times with 20 μL of demineralized water (Milli-Q)
and gently dried under a low flow of nitrogen gas. AFM images were
acquired using a BioScope Catalyst (Bruker, US) in the soft tapping
mode using a silicon probe, NSC36 tip B with a force constant of 1.75
N/m (MikroMasch, Bulgaria). Images were captured with a resolution
of 512 × 512 (10 μm × 10 μm) pixels per image
at a scan rate of 0.2 to 0.5 Hz. AFM images were processed with the
scanning probe image processor (SPIP, Image Metrology, Denmark) and
the Nanoscope Analysis (Bruker, US) packages. Fibril morphology was
analyzed using a custom fibril analysis Matlab script adapted from
the FiberApp package.53 (link)
five times diluted aggregated sample (initial αS concentration
= 50 μM and N-protein concentration = 0.2–1 μM)
was deposited onto freshly cleaved mica (Muscovite mica, V-1quality,
EMS, US) and left to rest for 5 min. Then, the sample was carefully
washed four times with 20 μL of demineralized water (Milli-Q)
and gently dried under a low flow of nitrogen gas. AFM images were
acquired using a BioScope Catalyst (Bruker, US) in the soft tapping
mode using a silicon probe, NSC36 tip B with a force constant of 1.75
N/m (MikroMasch, Bulgaria). Images were captured with a resolution
of 512 × 512 (10 μm × 10 μm) pixels per image
at a scan rate of 0.2 to 0.5 Hz. AFM images were processed with the
scanning probe image processor (SPIP, Image Metrology, Denmark) and
the Nanoscope Analysis (Bruker, US) packages. Fibril morphology was
analyzed using a custom fibril analysis Matlab script adapted from
the FiberApp package.53 (link)
10
Nanomechanical Characterization of Chondroprogenitors
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Chondroprogenitor cell monolayer cultures were imaged and analyzed using quantitative nanomechanical mapping (QNM) and force volume (FV) to assess changes in morphological and nanomechanical phenotype between the different populations at expansion stage and under differentiation conditions. Imaging experiments were performed using a Bioscope Catalyst (Bruker) instrument in either QNM or FV mode. High aspect ratio silicon probes, MLCT-D (Bruker), were used for the experiments with a spring constant of 0.03 N/m and a cantilever calibrated using in house manufacturer protocols before each experiment. Cells were imaged alive in DMEM without phenol red (Gibco) media, at 37°C. Care was taken to avoid the generation of imaging artefacts throughout. At least 6 cells per sample were analyzed taking 25 FV curves per cell in a 2.5 μm2 area in 30 min not to lose the phenotype or adherence of the cells. A 1 μm ramp size was applied to the sample with a force of 500 pN, the ramp rate was 1.03 Hz with constant forward and reverse tip velocity of 2.06 μm/s. When imaging in QNM mode, peak force amplitude and frequency were set at 500 nm and 0.25 kHz, respectively. The scan rate was 0.5 Hz and the 5.05 μm/s tip velocity, applying a force between 1 and 0.5 nN with feedback gain of 1.000.
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