Tapping mode AFM using the Bruker BioScope Catalyst Atomic Force coupled with Zeiss LSM5 Confocal Fluorescence Microscope was used on the cells at 37°C in cell culture media. AFM deflection images of cells were used in the imaging experiment. In the force measurement, sharp silicon nitride AFM probes (tip radius, 20 nm) were employed (Bruker Corp., USA). The spring constants of AFM tips were calibrated to be 0.10–0.11 N m−1 and deflection sensitivities were 45–50 nm V−1, using Thermo K Calibration (Agilent Technologies, USA). The approaching/retracting speed of the AFM tip during the force curve measurement was 6 μm s−1. Force–distance curves were recorded to obtain cell elasticity (Young’s modulus, E) of individual cells. For each time point, at least 20 single cells, 20 cells at the base of the iFA and 20 cells at the tip of the scar were measured with over 15 force–distance curves per cell to obtain significant results. The Young’s modulus was calculated via the Scanning Probe Image Processor (SPIP) software (Image Metrology, Denmark) by converting the force–distance curves to force–separation curves and fitting the Sneddon variation of the Hertz model, which describes conical tips indenting elastic samples.
Lsm5 confocal fluorescence microscope
Manufactured by Zeiss
The LSM5 Confocal Fluorescence Microscope is a high-performance imaging system designed for advanced fluorescence microscopy applications. It utilizes laser excitation and confocal principles to capture detailed, high-resolution images of fluorescently labeled samples.
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Lab products found in correlation
3 protocols using lsm5 confocal fluorescence microscope
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Cell Elasticity Profiling with Tapping Mode AFM
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Cell Elasticity Profiling with Tapping Mode AFM
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Quantifying Nuclear Stiffness by AFM
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SMCs in 35 mm glass coverslip-bottom dishes (World Precision Instruments) were incubated with Dox for 24 hours to induce nuclear lamin expression. Nuclear stiffness (Young’s modulus) in adherent cells was measured on a NanoWizard 4a Bioscience AFM (JPK Instruments) coupled with a Zeiss LSM5 confocal fluorescence microscope (53 (link), 54 (link)). A spherical AFM tip with a radius of 500 nm and 0.2 N/m spring constant cantilever (Nanotools, B500-CONT) was placed on top of the cell over the nucleus, identified by staining DNA with Hoechst 33342. Three different areas of the nucleus were sampled with a 2-nN maximum set point. The 3 measurements were used to calculate the average Young’s modulus for a single cell. All measurements were performed at 37° C in HEPES-buffered culture medium. The Young’s modulus was measured in 25 randomly selected cells for each group, and the force curves analyzed with JPK Data Processing software (JPK Instruments). The Young’s modulus was calculated with the Hertz model for a spherical tip and applied to fit the slopes of the approach curve.
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