Ntegra prima setup

For atomic force microscopy, fibrillar insulin was deposited on freshly cleaved mica and left to settle for 5 min, after which the mica plates were rinsed with milli-Q water and dried under a gentle stream of nitrogen. AFM images were recorded on an NTEGRA Prima setup (NT-MDT, Moscow, Russian Federation) equipped with a gold-coated single crystal silicon cantilever (NSG-01, spring constant ~ 5.1 N/m, resonance frequency of ~ 150 kHz). The images were processed in the Gwyddion software package (Nečas and Klapetek 2012 (link)) using planar subtraction, polynomial background subtraction and correction for linear aberrations. For fluorescence microscopy, 150 µl of insulin solution was placed on positively charged glass (Thermo Scientific, Waltham, MA, USA) and sealed with a 18 × 18 mm coverslip. A Zeiss AxioObserver.Z1 inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) with a 100 × oil immersion objective (NA = 1.46) was used in combination with a Photometrics Evolve EMCCD camera (Photometrics, Tuscon, AZ, USA), 100 ms exposure time, to obtain the images of the samples.

Samples from the ThT aggregation assays at 90 h were diluted 10 times in MilliQ water and deposited onto freshly cleaved mica. After 15 min, the mica samples were rinsed twice with MilliQ water then dried completely under a gentle stream of nitrogen. The images were captured using an NTEGRA Prima setup (NT-MDT, Moscow, Russia) at a resonance frequency around 180 kHz, using a gold-coated single crystal silicon cantilever (NSG01, spring constant of ~5.1 N/m; NT-MDT, Moscow, Russia). Images of 512 pixels were captured at a scan rate ranging from 0.3–0.5 Hz, scanning a minimum of four 50 µm × 50 µm areas in each sample. Images were analyzed using WSxM 5.0 software [60 (link)], with around 60 fibers measured per condition. The images shown in this article are representative for each condition.

Aggregated samples from ThT experiments were diluted into MilliQ water (10–20 times) and deposited on freshly cleaved mica. After 10 min, the mica was rinsed with filtered Milli-Q water and dried under a gentle nitrogen stream. Images were recorded on an NTEGRA Prima setup (NT-MDT) using a gold-coated single crystal silicon cantilever (NT-MDT, NSG01, spring constant of ~ 5.1 N/m) and a resonance frequency of ~ 180 kHz. 256 pixel images were acquired with 0.5 Hz scan rate. Images were analyzed using the WSxM 5.0 software (Horcas et al. 2007 (link)). For each sample images were collected in at least three different 50 × 50 µm areas, the images shown are representative for each sample.