Atomic force microscopy (AFM) images of implant materials were acquired using a JPK NanoWizard IV (Berlin, Germany) integrated with an up-right microscope (JPK “TopViewOptics” with Navitar long working distance zoom lens). Measurements were performed in ambient conditions and in intermittent contact mode, or AC mode, using uncoated silicon ACL cantilevers from AppNano (Mountain View, CA, USA) with typical resonance frequencies of 160–225 kHz, a spring constant of 36—90 N/m and average nominal tip radius of 6 nm. Scan speeds ranged between 0.2 and 0.4 Hz and total scan areas of 100 μm×100 μm were imaged per measurement to obtain surface parameters. Scans of 5 μm×5 μm were taken to obtain more detailed images of implant material and biofilm surfaces. Images were made at 5 random locations per disk for a total of N = 15 images per material and per timepoint. Surface roughness (Ra) in nm, as well as peak-to-valley roughness (Rt) or total height difference in nm were determined (Box 1 ). The captured images were processed using JPKSPM Data Processing software version 6.1.191 (JPK BioAFM, Bruker Nano GmbH, Berlin, Germany).
Jpk bioafm
Manufactured by Bruker
Sourced in Germany
The JPK BioAFM is a high-performance atomic force microscope (AFM) designed for biological applications. It enables researchers to study the structure and dynamics of biological samples at the nanoscale. The JPK BioAFM provides precise topographical and mechanical measurements of living cells, biomolecules, and other soft matter samples.
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4 protocols using jpk bioafm
1
Atomic Force Microscopy Characterization of Implant Materials
2
Hydrogel Bead Characterization by AFM
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Samples for the Atomic Force Microscopy (AFM) measurement were prepared by putting hydrogel beads with DMEM with supplements into a petri dish and letting them adsorb for 15 min. AFM images of the hydrogel beads were obtained with a NanoWizard 4 (JPK BioAFM, Bruker Nano GmbH, Berlin) using Quantitative Imaging measuring mode. The measurements were done at 21 °C in DMEM. To average over a more extensive area in the image for the hydrogel beads, a nanotools biosphere B2000-cont cantilever (nanotools GmbH, Munich) with a nominal spring constant of 0.2 N m−1 and a spherical tip with a nominal tip radius of 2 µm was used. As maximum indentation force, 0.3 nN was set. The cantilever approach and retract speed were 35 µm s−1 with a sample rate of 100 kHz. The resolution was set to 1.56 µm Pixel−1. Young’s modulus was obtained by analyzing the data with the JPK Data Processing Software (JPK BioAFM, Bruker Nano GmbH, Berlin, Germany). The images were bicubic interpolated.
3
Measuring Cell Mechanical Properties via AFM
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To analyse the mechanical properties of the cells, AFM was used. The cells were trypsinized right before the experiment and seeded in a confocal imaging dish (VWR) to a density of 500,000 cells/mL. To prevent any changes in mechanical properties due to formation of stress fibres (involved in cell attachment), the mechanical properties were measured immediately after adding the cells to the dish (while still in suspension). The AFM instrument is a Nanowizard 4 BioAFM (JPK bioAFM, Bruker). The measurements were performed in a liquid environment using an in‐house‐made colloidal probe with the radius of 5 μm. To extract the Young's modulus, the obtained force curves were processed in the JPK DP software using an adjusted Hertz model for spherical probes.
4
AFM Characterization of Macrophage Phagocytosis
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The atomic force microscopy (AFM) instrument used during all experiments is the Nanowizard 4™ with an automated stage (JPK BioAFM, Bruker). All force curves for Young's modulus measurements were acquired in contact mode using a colloidal probe, containing a spherical tip of 5 µm diameter (CP-qi-SCONT-BSG, force constant 0.1 N m -1 ) and using a setpoint of 2 nN at 2 µm s -1 . Viscoelastic properties were measured using the same AFM device equipped with a pyramidal tipped probe having a nominal radius of curvature of 10 nm (Force constant 0.3 N m -1 , DNP-S10; Bruker). During the viscoelasticity experiments, the cantilever oscillated with an amplitude of 50 nm at increasing frequencies (more specifically: 10,50,100,120,150 and 200 Hz). Processing of all AFM data was performed using JPK-Bruker software. finally, analysed by flow cytometry on the BD LSRII. Analysis was performed via FlowJo (v.10.8.1) software. The gating strategy was as follows: creating 'not debris' population from FSC x SSC, deriving F4/80+ population (representing BMDMs) from 'not debris' population, deriving CMFDA+ population from F4/80+ population (representing uptake of CMFDAstained MCA205 cells by BMDMs).
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