We performed the AFM measurement with BioScope Resolve system (Bruker, Billerica, MA, USA). A silicon nitride probe with a tip radius of 70 nm was used (PFQNM-LC-A-CAL, Bruker). The operation mode is PeakForce QNM in Fluid, and the pre-calibrated spring constant is 0.075 N/m. The AFM image was measured through NanoScope Analysis software. Briefly, the keratocytes were labeled by CellTracker CMFDA and then fixed in 4% formaldehyde. We first performed epifluorescence imaging of the cell to obtain the lamellipodium structure and cell migration mode. Then we performed AFM imaging of the cell, with a resolution at 64 × 64 and a line scan rate of 0.2 Hz. After flattening the AFM image of the cell by using the plane fit tool, the height profile of the cell lamellipodium was measured by using the section tool in the analysis software.
Pfqnm lc a cal
Manufactured by Bruker
Sourced in United States
The PFQNM-LC-A-CAL is a specialized lab equipment designed for precise material characterization. It functions as a Calibration Standard for Peakforce Quantitative Nanomechanical (PFQNM) measurements. The core purpose of this product is to provide a standardized reference for calibrating PFQNM-based atomic force microscopy (AFM) systems.
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Lab products found in correlation
Bioscope resolve system, by Bruker (1 mentions)
Cat no p35g 1.5 14 c, by MatTek (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Bioscope resolve, by Bruker (1 mentions)
Fv3000, by Olympus (1 mentions)
Nanowizard 3, by Zeiss (1 mentions)
Bioscope resolve afm, by Bruker (1 mentions)
Nanoscope analysis software, by Bruker (1 mentions)
6 protocols using pfqnm lc a cal
1
AFM Imaging of Keratocyte Lamellipodium
2
Measuring Tracheal Epithelial Cell Stiffness
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WT and Vangl2Lp/+ tracheal epithelial cells were seeded onto a glass bottom dish (Mattek, Cat. no. P35G-1.5–14-C) coated with collagen. At 72 h after seeding, cells were taken to NanoWizard 4 (JPK Bruker, Berlin, Germany) to measure stiffness of cells. Cone-tipped cantilevers (Bruker, PFQNM-LC-A-CAL) with spring constant 0.109 and sensitivity 10.42 nm/V were used at setpoint 0.2 nN for scanning using the Qi™ mode, with scan size ranging 5–30 μm with 256 × 256 pixels. Using JPK Data Processing software, Young’s modulus micrographs were generated and the stiffness of WT and Vangl2Lp/+ tracheal epithelial cells were compared. A total of eight to ten 0.55–1 μm2 areas in each cell periphery were averaged for each micrograph and data from 3-5 micrographs were averaged for each replicate experiment.
3
Measuring Cell Rigidity by AFM
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Force measurements were performed with the co‐axis system for confocal microscopy (FV3000, Olympus Corporation) and atomic force microscopy (BioScope Resolve, Bruker Corporation) as described (Kuo et al, 2021 (link)). Before measurements were made, the deflection sensitivity and spring constant of the atomic force microscopy probe (PFQNM‐LC‐A‐CAL, Bruker Corporation) were adjusted with the No‐Touch calibration mode. Briefly, living COS7 cells with microtubule disassembly and control COS7 cells were cultured in DEME with 10% FBS and located by confocal microscopy. The rigidity of the selected cell was measured with the Fast‐Force Volume mode at the indicated time point through atomic force microscopy. Then, cell rigidity was calculated from force‐distance curves that were fit to the Sneddon model through NanoScope Analysis. Cell rigidity at different time points was normalized to the original rigidity of that cell. In the contractile stress fiber inhibition experiment, transfected cells were pretreated with Blebbistatin (10 µM; Sigma Aldrich) for 2 h before AFM measurement.
4
Cantilever Force Calibration in Liquids
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Pre-calibrated peak force cantilevers (model PFQNM-LC-A-CAL; Bruker, Billerica, MA, USA) with a pre-defined nominal spring constant were used. A thermal of the cantilever was captured in deionized water to obtain the power spectral density (PSD) of the deflection data. The thermal was fitted with the single harmonic oscillator (SHO) or the Lorentzian model. The selected model was the Lorentzian one since it better fit the thermal acquired in liquids. For instrumental reasons, the data were captured with a deflection sensitivity based on an SHO fit, which was then corrected before analysis based on a Lorentzian fit (see Supplementary Materials, Figure S1 ) [16 (link)]. The Petri dishes with deionized water were fixed to an aluminum holder with vacuum grease and mounted on the atomic force microscope stage with two magnets. The inverse optical lever sensitivity (InvOLVS) of the cantilever was calculated using the no-touching calibration method. For frequency sweep, the hydrodynamic correction factor (b-hydrodynamics factor) of the cantilever was measured in each culture medium used following the procedure by Alcaraz et al. [17 (link)].
5
Atomic Force Microscopy of Cellular Elasticity
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Cells transfected with siRNA were either treated with CNF1 toxin for 2 h or detached and plated for 1 h 30 on 35 mm WilcoWell glass bottom dishes coated with fibronectin. AFM experiments were performed on a JPK NanoWizard III mounted on a Zeiss Observer Z1 optical microscope with temperature control. Before each experiment, cantilevers (precalibrated PFQNM-LC-A-CAL, Bruker) were calibrated according to the SNAP procedure111 (link) to eliminate any error in the deflection sensitivity determination. Each experiment was carried out on the same day, with the same cell culture and the same cantilever to account for slight variation in the tip shape manufacture, and was repeated at least three times. Cells were scanned in the perinuclear region in force distance mode (QI) with the following parameters: 300 pN force threshold and 50 µm/s tip velocity on a 40⨉40 (Fig. 5 ) or 20⨉20 (Fig. 6 ), 6 µm2 surface. Cortical elasticity (Young’s modulus) was computed using in-house Python software (version 3.6.9) by fitting the first 50 nm of cell indentation with the Hertz model for a sphere of 65 nm radius.
6
Nanomechanical Characterization of Cells
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AFM force spectroscopy experiments were performed with the operation mode of PeakForce QNM in Fluid available on a commercial AFM BioScope Re¬solve (Bruker, Billerica, MA, USA). Force mappings were obtained using a silicon nitride probe (PFQNM-LC-A-CAL, Bruker) with pre-calibrated spring constant of 0.091 N/m, tip height of 17 μm, and tip radius of 65 nm. Deflection sensitivity of the cantilever was calculated by thermal tune. Force curves were captured by Force Volume mode with scan size of 500 nm, ramps/line of 4, ramp rate of 1 Hz, ramp size of 1.5 μm and deflection error trigger threshold of 6 nm. All force curves were treated with Sneddon model in Nanoscope Analysis software (Bruker) to extrapolate the apparent Young's modulus with tip half angle of 18° and sample possion's ratio of 0.5. All experiments were done at room temperature in ECM within 1 h. Above16 force mappings were obtained per sample, and 30 different samples were analyzed for each condition.
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