The mechanical properties of individual ASCs, CMMPs, thin gels, and spheroids were characterized with an MFP-3D-BIO AFM (Asylum Research, Santa Barbara, CA) using previously described methods.8 (link) Samples were attached to plasma treated coverslips in 50 mm, low-profile Petri dishes with a thirty-minute incubation at 37°C and then gently flooded with 3 mL of phosphate buffered saline (PBS) or DMEM/F-12 for non-biological and biological samples, respectively. Elastic and viscoelastic properties were obtained from force vs. indentation/time curves using a modified, thin-layer Hertz model.7 (link), 9 (link) Spheroid heights were determined from the difference in z-position between the initial contact when positioned over the apex of the spheroid and over the glass adjacent to where each spheroid was adhered. Data were acquired using the settings specified in Table III .
Mfp 3d bio afm
Manufactured by Oxford Instruments
Sourced in United States
The MFP-3D-BIO AFM is an atomic force microscope designed for biological applications. It provides high-resolution imaging and analysis capabilities for studying biological samples at the nanoscale level.
Automatically generated - may contain errors
Lab products found in correlation
Pointprobe ppp fmr probes, by Nanosensors (2 mentions)
Tr400 pb cantilevers, by Olympus (2 mentions)
Eclipse ti u, by Nikon (2 mentions)
Ppp fmr, by Nanosensors (2 mentions)
Nsc35 c, by MikroMasch (2 mentions)
Fluorodish, by World Precision Instruments (2 mentions)
Dmem n2 medium, by Thermo Fisher Scientific (2 mentions)
Ld plan neofluar 5x 0.15na objective, by Zeiss (2 mentions)
Celltak tissue adhesive, by Corning (2 mentions)
Axio observer z1 inverted microscope, by Zeiss (2 mentions)
Igor software, by Oxford Instruments (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Falcon petri dishes, by BD (1 mentions)
Omcl tr400psahw, by Olympus (1 mentions)
Matlab, by MathWorks (1 mentions)
Axio observer, by Zeiss (1 mentions)
Metamorph, by Molecular Devices (1 mentions)
Glutaraldehyde solution, by Merck Group (1 mentions)
Igor pro 6.34a, by Oxford Instruments (1 mentions)
Mlct probe, by Bruker (1 mentions)
33 protocols using mfp 3d bio afm
1
Mechanical Characterization of Stem Cell Constructs
2
Imaging TRF2 and TIN2 Protein Interactions
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TRF2 (80 nM dimer) without or with TIN2 (TIN2S or TIN2L, 80, 120 and 200 nM) was incubated with the linear pT270 DNA (10.5 nM) in TRF2 Imaging Buffer (20 mM HEPES [pH 7.5], 100 mM NaCl) at room temperature for 10 min. All DNA–protein samples were diluted 10-fold in TRF2 Imaging Buffer before being deposited on to a freshly prepared 1-(3-aminopropyl)silatrane (APS)-treated mica (SPI Supply) surface for 30 s (56 (link)). The APS-treated mica surface was washed with DI water and dried under a stream of nitrogen gas. All images were collected in the tapping mode using Pointprobe® PPP-FMR probes (Nanosensors, spring constants at ∼2.8 N/m) on an MFP-3D-Bio AFM (Asylum Research). All images were captured at a scan rate of 1–2 Hz, a scan size of 1–3 μm × 1–3 μm and a resolution of 512 × 512 pixels.
3
EGFR-Chip Binding Force Characterization
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The topography and microstructure of the chip was obtained by MFP-3D-BIO AFM (Asylum Research, California, USA) with tapping mode. The binding force between the test sample and EGFR was measured on AFM by contact mode in PBS at room temperature. The location of immobilized EGFR on the chip was identified to allow tip moving on for later measurement. Stress-strain curve (force-distance curve) was obtained by moving the surface-modified tip to the EGFR-immobilized location, holding it on for several seconds to allow binding to occur and then retracting. All the measurements were executed at the same loading rate. The spring constant of cantilever was determined in air (measured values from 0.09 to 0.18 N/m). Curves showing significant non-specific interactions as well as those showing a zero interaction were not analyzed.
4
Determination of Collagen Stiffness via AFM
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A commercial atomic force microscope (MFP-3D-BIO AFM, Asylum Research) was used for the determination of the elastic moduli (i.e., stiffness) as previously described (Staunton et al., 2016 (link); Wu et al., 2018 (link)). Briefly, spheroconical probes (LRCH, Team Nanotech) with nominal spring constants of 0.2 N ⋅ m−1, a half cone angle of 18.8°, tip length of >10 µm, and a spherical radius of 700 nm were used to collect force–indentation curves in four to seven 4 × 5 grids of 90 × 90-µm areas at 37°C in PBS buffer with an indenter vertical speed of 2 µm ⋅ s−1. Trigger forces of 20–30 nN resulted in indentation depths ≥10 µm. The elastic moduli were obtained by fitting the initial 10 µm of indentation of each force–indentation curve to a nonadhesive elastic contact model for spheroconical probes (Staunton et al., 2016 (link)). Collagen was assumed to be incompressible, with a Poisson ratio of 0.5 (Lacroix et al., 2018 ). The spring constants of the cantilevers were determined by the thermal noise method before the experiment (Butt and Manfred, 1995 ).
5
AFM Imaging and DREEM Analysis of mtSSB-DNA Complexes
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AFM imaging in air was done using the AC mode on a MFP-3D-Bio AFM (Asylum Research) and Pointprobe® PPP-FMR cantilevers (Nanosensors, spring constants at ∼2.8 N/m). All images were captured at a scan rate of 1–2 Hz and a resolution of 512 × 512 pixels. DREEM images were collected as described previously (32–34 (link),48 (link)). Briefly, the AFM cantilever and the bottom of the mica substrate were coated with a thin layer of colloidal liquid silver (Ted Pella Inc.). A function generator (Sanford Research System, model DS335) was used to generate the AC bias at the first overtone and DC bias that were applied between the AFM cantilever and mica substrate. A lock-in-amplifier (Sanford Research System, model SR844 RF) was used to monitor the changes in vibration amplitude and phase at the first overtone as a function of sample positions. To optimize DREEM signals, AC and DC bias were adjusted from 0 to 20 V and −1.5 to 1.5 V, respectively. AFM volumes (mean ± standard deviation) of mtSSB alone and mtSSB–DNA complexes were measured using Gwyddion software. The types of AFM cantilevers and imaging conditions in this study matched our previous experiments relating molecular weight and AFM volume of proteins (34 (link)).
6
Atomic Force Microscopy of Tumor Tissue
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Non-necrotic 10 to 12 week old tumors were gently dissected away from the skin and flash frozen in OCT. Tumors were sectioned at 20 μm per section. Just prior to AFM, tissues were quickly thawed in 1XPBS at room temperature and then maintained in 1X PBS supplemented with protease inhibitor cocktail (Roche Diagnostics, 11836170001) and propidium iodide (20 μg/ml). 5–6 force maps were taken of at least two tumors from three mice per group. AFM was performed as described (Acerbi et al., 2015 (link)). All indentations were taken on an MFP-3D-BIO AFM (Asylum Research) mounted on an Olympus X711 inverted fluorescent microscope in an TMC acoustic noise enclosure. We used silicon nitride cantilever tips with a 5 μm borosilicate glass sphere affixed to the tip with a spring constant of 0.06 N/m (Novascan, Boone, IA). The cantilever was calibrated with thermal oscillation prior to each experiment. Indentations were taken at 20 um/second loading-rate with a maximum force of 5 nN, and force maps were generated using the FMAP function on IGOR software (Asylum Research). The Hertz method was used to calculate elasticity and Poisson’s ratio of 0.5 was used to calculate Young’s elastic modulus.
7
Optimized AFM Protocol for Measuring EC Stiffness
8
Atomic Force Microscopy Analysis of MWCNT-Induced Cell Stiffness
9
Atomic Force Microscopy of Fixed Cells
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AFM imaging was performed using MFP 3D-BIO AFM (Asylum Research, Oxford Instruments) and utilizing cantilever OMCL TR400PSA HW (Olympus). Cells were cultured on glass coverslips (ϕ 24) for 24 h, then fixed with 4% formaldehyde in PBS and dried. Imaging was done in contact mode in the air. The scanning area was set to 90 µm, set point to 1 V, integral gain to 10, proportion gain to 0, scan rate 1 Hz, and 1024 scan points. Gwyddion software was used to analyze the obtained images.
10
Viscoelasticity of Tumor Spheroids
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Example 9
The elastic and viscoelastic properties of individual spheroids were characterized once a week for three weeks using an MFP-3D Bio AFM (Asylum Research) equipped with a 0.35 N/m silicon nitride cantilever tipped with a 25 μm polystyrene bead (Novascan Technologies, Inc., Boone, IA, PT.PS.SN.25). Once a week, fifteen spheroids for each microbead condition from both media environments were mechanically characterized. Spheroids cultured in each of adipogenic and control medium were tested on consecutive days for the first iteration of the example; this testing order was reversed for the second iteration. Tests were performed by positioning the cantilever over the center of each spheroid and performing a single indentation using a 10 μm/s approach velocity, 30-second relaxation period, and 30 nN trigger force. Data were analyzed using a custom MATLAB program.
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