Mfp 3d bio

Topographic imaging of cells by atomic force microscopy (AFM) was performed with either MFP-3D BIO (Asylum Research, Santa Barbara, CA) or BioScope SZ AFM (Bruker, Santa Barbara, CA). The MFP-3D BIO is integrated on an Olympus IX51 inverted microscope and the BioScope SZ on an Olympus IX71 (Shinjuku, Tokyo, Japan). Silicon nitride triangular cantilevers with a nominal spring constant of 0.02 N/m were used (TRP400PSA; Asylum Research, Santa Barbara, CA). Live cells were imaged in 2 mL of HBSS diluted in 20 mM of HEPES buffer at room temperature. Height and deflection images were acquired in contact mode scanning over 30 μm × 30 μm to 50 μm × 50 μm regions at scan rates between 0.2 and 0.5 Hz. Fixed cells were subsequently imaged by AFM. To fix the cells, the devices were incubated in 4% paraformaldehyde diluted in PBS for 20 minutes at room temperature and then replenished with PBS. Imaging of fixed cells was carried out with similar AFM scan parameters as live cells.
AFM image analysis was performed using the NanoScope Analysis Version 1.40r51 software (Santa Barbara, CA). Images were processed by low-pass filtering and flattening to remove noise. Simultaneous optical and AFM images were also obtained of the cell-loaded devices using the AFM-inverted microscopes.