All force data was collected with an Asylum MFP-3D Bio AFM (Asylum Research, Santa Barbara, CA). 5 µm polystyrene beads (Corpuscular, NY) were attached with Norland Optical Adhesive (Norland, NJ) to Arrow-TL1 tipless cantilevers (nominal k = 0.03 N/m, Nanoworld). Cantilever spring constants were calibrated using the thermal tuning method. The AFM cantilever was loaded into the AFM cantilever holder and 10 µL of 0.01 mg/mL fibronectin in PBS was placed on the end of the cantilever where the bead was attached. The solution was allowed at least 15 minutes to physisorb onto the polystyrene bead before rinsing with medium and loading into the AFM head.
Asylum mfp 3d bio afm
Manufactured by Oxford Instruments
Sourced in United States
The Asylum MFP-3D Bio AFM is a high-performance atomic force microscope designed for biological and soft matter applications. It provides accurate topographical and mechanical measurements at the nanoscale level.
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6 protocols using asylum mfp 3d bio afm
1
Fibronectin Adhesion Force Measurement
2
Characterization of Catecholamine Coatings
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Firstly, scanning electron microscopy (SEM, JSM-7800F, Electronics, Japan) was used to analyze the surface morphology of the catecholamine coatings. All the samples were dehydrated, dealcoholized, and dried before analysis. SEM was operated at 2.7 kV × 15 mA under a pressure of 5 × 10−4 Pa. Then the surface roughness of the coatings was measured via atomic force microscopy (AFM, Asylum MFP-3D-BioAFM, Asylum Research, USA) in a non-contact mode with Si cantilevers, in a scanning range of 5 μm × 5 μm. The thickness of the coatings was determined by using a spectroscopic ellipsometer (M − 2000V, J.A. Woollam, USA) using the Cauchy model. To analyze the water contact angle of the coatings, a Drop Shape Analysis System DSA100 (Krüss, Hamburg, Germany) was used, and the DSA 1.8 software was adopted to process the image of a sessile drop of 5 μL distilled water.
3
Transistor Characterization via AFM and SKPM
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The transistor characterization measurements were carried out in the dark and under ambient conditions using an Agilent 4155 C Semiconductor Parameter Analyzer. AFM and SKPM measurements were taken using an Asylum MFP-3D Bio AFM (Asylum Research, USA) in ambient atmosphere (the identification of commercial equipment or vendor is not intended to imply recommendation or endorsement by NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose). For AFM, a silicon cantilever (Nanosensors PPP-NCLR, force constant: 21–98 N m−1, resonance frequency: 146–236 kHz) was used in tapping mode with a feedback setpoint of 500 mV, and 1 µm × 1 µm images were taken at a rate of 0.5 Hz. SKPM measurements used a silicon cantilever with a Ti/Ir coating (Oxford Instruments ASYELEC.01-R2, force constant: 1.4–5.8 N m−1, resonance frequency: 58–97 kHz) at a nap height of 5 nm, and 20 µm × 20 µm images were taken at a rate of 1 Hz.
4
Quantifying Mammary Tissue Stiffness via AFM
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Mice were euthanized and a small piece of mammary tissue (5 mm x 5 mm) was excised from the nipple end of the #9 inguinal gland. This tissue was then attached to a coverslip using a biocompatible glue (epoxy resin) as described previously [35] (link). The tissue was then immediately immersed in sterile saline and atomic force microscopy performed. Briefly, stiffness measurements were performed using Asylum MFP-3D Bio AFM (Asylum Research, Goleta, CA, USA) mounted on an inverted fluorescent microscope (Nikon Eclipse TE2000-U). Samples were indented using silicon nitride cantilevers with a 10 μm polystyrene spherical tip and nominal spring constant of 0.01 N/m. Force maps at 5–10 regions of 20μmx20μm were collected and approximately 250–300 force-displacement curves were analyzed per sample for each matched pair (wild type and mutant) experiment. Force curves were analyzed using the Oliver-Pharr model to compute the tissue Young's modulus.
5
Characterizing DNA-Protein Complexes by AFM
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DNA-protein complexes were imaged using an Asylum MFP 3D Bio AFM (Oxford Instruments, Goleta CA) with an Olympus AC240TS probe in tapping mode at room temperature. The samples were prepared a suitable concentration (0.5–1.0 ng/μL), then 40 uL of prepared samples were slowly deposited to a freshly cleaved AP-mica for 5 minutes and rinsed with 1 mL ultrapure deionized water twice before being gently dried with UHP argon gas.
AFM images were collected at a speed of 0.5–1 Hz at 512 × 512-pixel resolution, with an image size of 2 μm. For analysis, raw images were exported into 8-bit grayscale Tiff images using the Asylum Research’s Igor Pro software and imported into FIJI/ImageJ (NIH) for detection of single particles and quantification of volume, surface area, and volume/surface area ratio using as has been done previously for studies of chromatin compaction via AFM23 . In order to assess single chromatin particles, rather than potential clusters of multiple fibers or residual MMTV mononucleosomes, only particles with volumes measuring between 2,000 and 15,000 nm3 were included in analyses.
AFM images were collected at a speed of 0.5–1 Hz at 512 × 512-pixel resolution, with an image size of 2 μm. For analysis, raw images were exported into 8-bit grayscale Tiff images using the Asylum Research’s Igor Pro software and imported into FIJI/ImageJ (NIH) for detection of single particles and quantification of volume, surface area, and volume/surface area ratio using as has been done previously for studies of chromatin compaction via AFM23 . In order to assess single chromatin particles, rather than potential clusters of multiple fibers or residual MMTV mononucleosomes, only particles with volumes measuring between 2,000 and 15,000 nm3 were included in analyses.
6
Tapping Mode AFM Imaging in Liquid
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All AFM images were acquired in liquid (DI water) with tapping mode on an Asylum MFP-3D-Bio AFM (Oxford Instruments-Asylum Research, Santa Barbara, USA). Tapping mode cantilevers with a resonant frequency of 70 kHz were used (240AC-PP manufactured by NANOANDMORE USA). Images were acquired by oscillating the cantilevers at their 1st resonant frequencies and parameters were optimized so that very minimal force was applied to avoid tip-induced sample modifications. The resulting images were then processed and the RMS roughness value extracted in Gwyddion software (post-2.52 development version) (Nečas, 2012 (link)).
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