Nanoscope analysis

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.

AFM imaging was performed on a Dimension FastScan atomic force microscope (Bruker, Santa Barbara, CA USA) in PeakForce Tapping^TM Mode using ScanAsyst-Air probes (nominal spring constant: 0.4 N/m and tip radius: 5 nm, Bruker) for fixed and air-dried samples. Sample imaged under fluid conditions were imaged in 0.22-μm-filtered autoclaved seawater with ScanAsyst-Fluid probes (nominal spring constant: 0.7 N/m and tip radius: 20 nm, Bruker). Acquired AFM micrographs were processed and analyzed using Nanoscope Analysis (Bruker) and Gwyddion software (http://gwyddion.net). Raw AFM height image data, generated from the feedback-controlled scanning tip motion, was processed using minimal line-flattening and plane-fitting routines. Peak force error image data, generated from the setpoint error of the applied peak forces by the scanning tip, were minimally processed using low-pass filters. Select AFM images were processed to improve visualization of surface features using Gwyddion software to generate SEM image presentations from height data simulated by Monte Carlo integration [61 ].

Pistils were placed straight in a 2% agar MS medium and 0.8% low-melting agarose was added up to a certain level where the papilla cells were maintained well immobilised in agarose while leaving the top accessible to the indenter. This set up allowed accurate measurements of cell wall stiffness on the dome-shaped top of papilla cells. AFM indentation experiments were carried out with a Catalyst Bioscope (Bruker Nano Surface, Santa Barbara, CA, USA) that was mounted on an optical microscope (MacroFluo, Leica, Germany) equipped with a x10 objective. All quantitative measurements were performed using standard pyramidal tips (RFESP-190 (Bruker)). The tip radius given by the manufacturer was 8–12 nm. The spring constant of the cantilever was measured using the thermal tune method and was 35 N/m. The deflection sensitivity of the cantilever was calibrated against a sapphire wafer. All experiments were made in ambient air at room temperature. Matrix of 10 × 10 measurements (step 500 nm) was obtained for each papilla, with a 1µN force. The Young’s Modulus was estimated using the Nanoscope Analysis (Bruker) software, using the Sneddon model with a < 200 nm indentation.