Msnl cantilevers

Atomic force microscopy (AFM) was employed to record the topography of treated bacterial cells for the qualitative assessment of AuNPs-mediated bacterial treatment. Representative strains of E. coli and P. aeruginosa were resuspended in distilled water (OD₆₀₀ ~ 0.1) and incubated with indicated concentrations of AuR NPs at 37 °C for 1 h. Then, 200 μL bacterial samples were transferred to the mica surface previously functionalized with 0.5% APTES. The attachment of bacterial cells to the mica surface was achieved during 20 min of incubation. Images of bacterial cell surface were collected using a Nano Wizard 4 BioScience AFM (JPK Instruments, Berlin, Germany) operated in Quantitative Imaging Mode. MSNL cantilevers (Bruker, MA, USA) with a nominal spring constant equal to 0.1 N/m were employed. The bacterial cells were located using an optical microscope to collect the topography and adhesion images. Then, 10 μm × 10 μm scanning was performed with the resolution of 128 pixels per line.

Topological images (10 × 10 μm²) of the tissue were acquired on the unfixed sections by Atomic Force Microscopes (AFM Nanowizard I and III, Bruker-JPK, Berlin–Germany) operated in contact mode (MSNL cantilevers (Nom. tip radius: 2 nm, triangular geometry, Bruker, Santa Barbara) operated in ambient conditions at a scanning rate of 1.0 Hz or above. To avoid bias in selecting the area to be imaged, we used a random walk approach to land the probe in each dermal layer by using the AFM sample holders’ x-y translation screws to move the sample (within each dermal layer). Images were then acquired and optimized where the probe came into contact with the sample regardless of the topology seen in the image acquired. A total of 14 images were acquired for each donor (papillary dermis 7; reticular dermis: 7 on a minimum of 2 sections per donor), leading to a dataset of 420 (10 × 10 μm²) images. Following their acquisition, each AFM image was plane-fitted before being segmented into 100 x (1 × 1 μm²) images using ImageJ, creating a final topology dataset of 42,000 (1 × 1 μm²) images to be analyzed.