MCF-7 cells were transfected with plasmid DNA encoding GFP-tagged NMHC-IIA, -IIB, or -IIC1. Culture dishes were mounted onto the stage of an Asylum MFP3D atomic force microscope (Asylum Research) coupled to a Zeiss epifluorescence microscope and indented using a pyramid-tipped probe (Olympus) with nominal spring constant of 20 pN/nm. GFP-positive cells were selected for the measurements, which were done in a stretch of the membrane where there was no protrusion. Actual spring constants were estimated using a thermal calibration method, and force curves were recorded for >20 cells for each sample. Force–indentation profiles were fit with a modified Hertzian model of a cone indenting a semi-infinite elastic material to extract the magnitude of cortical stiffness (MacKay and Kumar, 2013 (link)).
Asylum mfp 3d atomic force microscope
Manufactured by Oxford Instruments
Sourced in Japan
The Asylum MFP-3D is an atomic force microscope (AFM) designed for high-resolution imaging and analysis of surface topography and properties at the nanoscale. It provides precise three-dimensional mapping of sample surfaces with sub-nanometer resolution. The MFP-3D is a versatile instrument capable of operating in various imaging modes to investigate a wide range of materials and samples.
Automatically generated - may contain errors
Lab products found in correlation
Tm 1000 sem, by Hitachi (2 mentions)
Igor pro 6.22a, by Wavemetrics (2 mentions)
Pyramid tipped probe, by Olympus (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Zen blue 3, by Zeiss (1 mentions)
Axio imager m2 optical microscope, by Zeiss (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using asylum mfp 3d atomic force microscope
1
Measuring Cortical Stiffness in GFP-tagged Cells
2
AFM Imaging of Molecular Complexes
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Complexes were prepared as described above and deposited on a piece of freshly cleaved mica. The solution was incubated for 5 minutes on the mica surface for complete attachment. Excess solution was removed with a gentle stream of nitrogen gas and the sample was imaged immediately in AC mode by an Asylum MFP-3D atomic force microscope (Asylum Research, Santa Barbara, CA, USA), equipped with a Multi-75Al tip.
3
Quantifying Hydrogel Indentation Moduli
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The indentation moduli of hydrogels were quantified using an Asylum MFP3D atomic force microscope (Asylum Research, CA). We used polystyrene colloidal probe tips with radius R ∼ 12.5 μm (Polysciences, PA) attached to tip-less cantilevers with nominal spring constants of k ∼ 0.12 N/m (Bruker, CA). The colloidal probes were attached to the cantilever via lift-off process39 (link). For each probe tip, the exact spring constants of the cantilevers were directly measured using thermal calibration method58 . The relationship between the detected voltage and the applied force was calibrated by bringing the cantilever in contact with a glass slide and calculating the slope of the voltage-displacement curve. The displacement, d, was translated to force, F, using Hooke’s Law (F = kd). The indentation was performed under a force control scheme (max force ~20 nN), limiting the indentation depths to 0.5–3 μm. Each hydrogel was indented at 8–10 locations with 100 um spacing. The Hertz model was fit to the force-displacement curve to obtain the indentation modulus, E. An indentation velocity of 2 μm/s ensured evaluating the indentation modulus at close to equilibrium condition.
4
Surface Roughness and Wettability Characterization
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80 × 80 μm areas of PEEK and PS discs were probed for surface roughness using an Asylum MFP-3D atomic force microscope (AFM, Asylum Research, Oxford Instruments) operated in tapping mode in air. Root mean square (RMS) roughness was calculated from topographic images after plane subtraction and drift correction. Four samples of each surface type were assessed. For visualization with scanning electron microscopy (SEM), surfaces with biofilms were fixed with 4% paraformaldehyde, 1 h; dehydrated with an ethanol series, air-dried overnight, sputter-coated with gold, and visualized using a Hitachi TM-1000 SEM (Hitachi High-Technologies). For each condition, at least 3 independent surfaces were assessed.
Rough contact angles were measured on dry surfaces or after pre-incubation in SynF, or TSB for 1 hr at room temperature. Perpendicular views of droplets formed with 10 μL of distilled water were recorded digitally and angles measured using two methods – (1) direct measurement of the angle of the intersection with the surface of (2) measurement of the heights and the radii of the drops, followed by calculation of the angles from their ratios.
Rough contact angles were measured on dry surfaces or after pre-incubation in SynF, or TSB for 1 hr at room temperature. Perpendicular views of droplets formed with 10 μL of distilled water were recorded digitally and angles measured using two methods – (1) direct measurement of the angle of the intersection with the surface of (2) measurement of the heights and the radii of the drops, followed by calculation of the angles from their ratios.
5
Surface Roughness and Wettability Characterization
6
Multimodal Analysis of Bacterial Morphology
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Bacterial cell morphology was analyzed using a combined scanning system consisting of an Olympus FV 1000 confocal laser scanning microscope (CLSM) (Olympus Corporation, Tokyo, Japan) and an Asylum MFP-3D atomic force microscope (AFM) (Asylum Research, Santa Barbara, CA, USA). AFM scanning was performed in a semi-contact mode in air using an AC240TS silicon cantilever with resonance frequency of 50–90 kHz and contact stiffness of 0.5–4.4 N/m. Images were analyzed using the FV10-ASW 3.1 program (Olympus Corporation, Tokyo, Japan). To obtain the combined AFM-CLSM images, the CLSM image was imported into the AFM software (Igor Pro 6/22A Wave Metrics, Portland, OR, USA).
7
Multimodal Bacterial Cell Imaging
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Cells were visualized using an Axio Imager M2 optical microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) in phase-contrast and fluorescent modes. The images were captured using cameras such as Axoicam 506 Color and Zen Blue 3.1 (Carl Zeiss Microscopy GmbH, Jena, Germany). The morphometric parameters of bacterial cells were investigated using a unique combined scanning system consisting of an Olympus FV 1000 confocal laser scanning microscope (CLSM) (Olympus Corporation, Tokyo, Japan) and an Asylum MFP-3D atomic force microscope (AFM) (Asylum Research, Santa Barbara, CA, USA). Sample preparation and AFM scanning procedure were carried out according to Kuyukina et al. [31 (link)]. For that purpose, the cell suspension (15–20 µL) was stained with a two-component fluorescent dye LIVE/DEAD®BacLightTM Bacterial Viability Kit (Invitrogen, Carlsbad, CA, USA). AFM scanning of the preparations was performed in a semi-contact mode in air at frequency of 0.2 Hz using an AC240TS silicon cantilever with resonance frequency of 50–90 kHz, spring constant of 0.5−4.4 N/m, and the curvature radius of the probe at 9 nm. The root-mean-square surface roughness and dimensions (length and width) of living cells were calculated using the Igor Pro 6.22A (WaveMetrics, Portland, OR, USA) software. Cell volume and surface area were calculated using the formulas in [32 (link)].
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