Multimode 8 microscope

Exposed and as-cast blend films of E:[70]PCBM and ET:[70]PCBM on glass are investigated for morphological differences in ScanAsyst mode on a Bruker Multimode 8 microscope (Model number: MMAFM-2) with ScanAsyst-Air probes (spring constant: 0.4 N/m, resonant frequency: 70 kHz, nominal tip radius: 2 nm). All samples are scanned at 5 µm, 1 µm, and 500 nm at a scan rate of 0.8 Hz and a resolution of 640 samples per line. Both height and peak force errors are collected for analysis during the scan. We used NanoScope Analysis (provided by Bruker) for data analysis and converting 2D morphology into the 3D structure for better interpretation.

All AFM imaging was performed in liquid using Peakforce Tapping with either a Dimension FastScan microscope (images presented in Figs 1A and B, and 2A and D, and EV1) or a Multimode 8 (images presented in Fig 1C). FastScan D cantilevers (Bruker) were used with the Dimension FastScan microscope and PeakForce HIRS‐F‐B cantilevers (Bruker) were used with the Multimode 8 microscope. FastScan D cantilevers were operated using a drive frequency of 8 kHz and PeakForce HIRS‐F‐B cantilevers were operated at 4 kHz drive frequency. The areas imaged were 0.8 × 0.25–4 × 2 µm² in size and recorded at 3.5 Hz (FastScan) or 1.75 Hz (Multimode 8) line rate. Images were 512 × 256, 512 × 172 or 384 × 172 pixels in size.
During AFM movie acquisition, disaggregation was initiated by retracting the cantilever tip ~100 nm from the surface and injecting 0.75–1 µM Hsc70, 0.37 – 0.5 µM DNAJB1 and 0.07–0.1 µM Apg2 (always at a Hsc70:DNAJB1:Apg2 molar ratio of 1:0.5:0.1) in disaggregation buffer (HKMD buffer for ‐ATP controls) into the sample droplet. AFM imaging was continued within 1 min of injection of chaperones. The same area of αSyn fibres was imaged for >2 h at either at room temperature or at 30°C.

Atomic force microscopy was performed on samples pretreated with TFA and then dissolved in the desired volume of PBS to obtain a concentration of 100 μm. Samples were then incubated at 37 °C without shaking, and images were acquired at different time points. Height peak force error images were acquired on a Bruker Multimode 8 microscope with a Nanoscope V controller (Bruker UK Ltd., Santa Barbara, CA) operating in peak force tapping mode using ScanAsyst Air cantilevers (115-μm nominal length, 25-μm nominal width, nominal spring constants of 0.4 newtons/m, and typical resonant frequencies of 70 kHz). The ScanAsyst probes have a 2-nm nominal tip radius of curvature. Image data were acquired at peak force frequency of 4 kHz and a line rate of 3 Hz at a resolution of 512 pixels/line. Samples were diluted 1:10 and 1:100, and 100 μl of each diluted solution was added onto freshly cleaved mica and incubated at room temperature for 5 min. The excess of liquid was dried off from the mica and rinsed extensively with a gentle flux of filtered Milli-Q®-H₂O.

The AFM analysis has been performed using a Multimode 8 microscope (Bruker, Santa Barbara, CA). Two central studies have been conducted in the sample:127-Pluronic-[²²³Ra] RaCl₂ nanomicelles. The morphology and topography of the nanomicelles were analyzed. For these measurements, Scanasyst Air probes were used, with a nominal tip ratio of 2 nm and nominal spring constant of 0.4 N/m. However, the actual spring constant was calibrated by the thermal noise method. A drop of the nanomicelles solution was deposited in freshly cleaved mica to form the nanomicelles film. The scanning mode used was Peak Force Tapping Quantitative Nanomechanics (QNM), with a resolution of 256 × 256 lines per scan and scan frequency of 0.5 Hz.

Atomic force microscopy images and nanomechanical data were collected using a Multimode 8 microscope (Bruker, USA) operating in PeakForce Tapping mode. The specimens were imaged in room condition using ScanAsyst-Air probes (Bruker) (nominal length 115 μm, tip radius 2 nm, spring constant 0.4 N m⁻¹) for topography and RTESP-300 probes (Brukes) (nominal length 125 μm, tip radius 12 nm, spring constant 40 N m⁻¹). Prior to imaging, the probes were calibrated according to the manufacturer’s protocol using the Bruker calibration kit. Images were collected in height sensor, peak force error, Young modulus (DMT model) and adhesion channels. The raw AFM data obtained were processed using Nanoscope Analysis v.1.7. software (Bruker).

Height and peak force error AFM images were obtained on a Bruker Multimode 8 microscope with a NanoScope V Controller (Bruker United Kingdom Ltd.). Images were acquired operating in peak force tapping mode using ScanAsyst Air cantilevers using ScanAsyst probes with a 2 nm nominal tip radius of curvature. Image data were obtained at a peak force frequency of 4 kHz and a line rate of 3 Hz, at a resolution of 512 pixels/line. 10 µl sample was loaded onto freshly cleaved mica and incubated for 10 min at ambient temperature. The liquid excess was dried off from the mica, which was rinsed three times with a gentle flux of 0.22 μm filtered Milli-Q water. Aggregated Tau35 was left on formvar/carbon-coated 400-mesh copper grids (Agar Scientific) for 60 s before blotting the excess using a filter paper. The grids were washed with 4 μl 0.22 μm filtered Milli-Q water and negatively stained with 4 μl 2% (w/v) uranyl acetate for 30 s. TEM images were collected using a JEOL JEM1400-Plus transmission electron microscope operated at 80 kV. Detection was achieved using a 4kx4K OneView camera (Gatan). Acquisitions were performed at 25 fps and automatically corrected for drift using DigitalMicrograph® software (GMS3, Gatan).