Nanoscope 3 controller

For the AFM measurement, 5 μl of Aβ₄₂ sample solutions were deposited onto a cleaved mica substrate for 10 min and were rinsed several times with DI water to remove remaining salts and unbound peptides. After they were fully dried, AFM images were acquired in a tapping mode with an NCHR silicon cantilever (Nanosensors Inc.) using a Multimode AFM instrument equipped with a Nanoscope III controller and “E”-type scanner (Digital Instruments Inc.).

The corneocytes adherent to the tape strips were analyzed by AFM as described [36 (link),45 (link)]. In brief, each third consecutive tape strip was subjected to AFM analysis carried out with a multimode atomic force microscope equipped with a Nanoscope III controller and software version 5.30 sr3 (Digital Instruments, Santa Barbara, California). Silicon-nitride tips on V-shaped gold-coated cantilevers were used (0.01 N/m, MLCT, VEECO, Mannheim, Germany). Imaging was performed in air at ambient temperature with forces less than 1 nN at one to three scan lines per second (1–3 Hz) with a resolution of 512 × 512 pixels. For nano-object analysis, sub-cellular scan areas of 20 × 20 μm² were recorded. Topographical data of the cell surfaces were analyzed using the nAnosticTM-method applying custom-built, proprietary algorithms (Serend-ip GmbH, Muenster, Germany). Nanostructures protruding from the mean surface level are referred to as CNOs. CNOs of size < 500 nm were counted, the average object count of 10 areas is referred to as DTI [36 (link),37 (link)].

Corneocytes from patients were analyzed with AFM, as previously described.²¹ (link) Briefly, in each case the seventh tape strip was subjected to AFM measurements carried out with a Multimode AFM equipped with the Nanoscope III controller and software version 5.30sr3 (Digital Instruments, Santa Barbara, Calif). Silicon-nitride tips on V-shaped gold-coated cantilevers were used (0.01 N/m, MLCT; VEECO, Mannheim, Germany). Imaging was performed at ambient temperature with forces of less than 1 nN and 1 to 3 scan lines per second (1-3 Hz) with 512 × 512 pixel resolution. For texture analysis, subcellular scan areas of 20 μm² were recorded. Ten random images were analyzed from each sample.
Topographic cell-surface data were analyzed with the nAnostic method, applying custom-built proprietary algorithms (Serend-ip GmbH, Munster, Germany). The principle of this method has been described elsewhere.²² (link) Briefly, each nanostructure protruding from the mean surface level was morphometrically evaluated. These objects were then filtered by size and shape through computer vision. At this stage, only structures of positive local deviational volume smaller than 500 nm in height and with an area of less than 1 μm² were considered. The DTI score is the count of identified objects per image (a mean value from 10 randomly recorded images).

Contact mode Atomic Force Microscopy (AFM) on cultivated cells was performed as described before [63 (link)]. In this study, cells were chemically stabilized by glutardialdehyde fixation (1% final concentration). Briefly, AFM measurements were carried out in PBS-buffered solution (pH 7.4) using a Multimode AFM equipped with Nanoscope III controller and software version 5.30 sr3 (Digital Instruments, Santa Barbara, CA, USA). Silicon-nitride tips on V-shaped gold-coated cantilevers were used (0.01 N/m, MLCT, VEECO, Mannheim, Germany). Imaging was performed at ambient temperature with forces <1 nN at 1–3 scan lines per second (1–3 Hz) with 512*512 pixels resolution. For texture analysis, subcellular scan areas of 10μm² are recorded. Topographical data of the cell surfaces were analyzed using the nAnostic™-method applying custom-built, proprietary algorithms (Serend-ip GmbH, Münster, Germany). The method principle has been described before [64 ]. Briefly, each nanostructure protruding from the mean surface level is morphometrically evaluated. Then, they are filtered by their size and shape through computer vision; here, only structures of positive local deviational volume (LDV), smaller than 10³ nm in height and an area smaller than 10⁶ nm were considered. Values are given for the average depth of such objects (per image) and the sum of their deviational volumes (LDVs).