Multimode 8

Blc. viridis photosynthetic membranes were immobilized on the mica substrate in 40 μl absorption buffer (10 mM Tris-HCl pH 7.2, 150 mM KCl, 25 mM MgCl₂) for 1 h at room temperature. The sample was rinsed with imaging buffer (10 mM Tris-HCl pH 7.2, 150 mM KCl). AFM imaging was performed using a Bruker Multimode 8.0 equipped with a 97 μm J-scanner and OTR4-10 probe (spring constant = 0.08 N m⁻¹) in PeakForce Quantitative Nanoscale Mechanical (PeakForce QNM) mode. Minimal loading forces of ~120 pN were used at scan frequencies of 3 Hz using optimized feedback parameters. High-speed AFM was carried out using a NanoWizard 3 AFM (JPK) equipped with an ULTRA S scanner and Ultra-Short Cantilever probe (0.3 N·m⁻¹, Nano World) in AC mode with the scan frequency of 20–30 Hz. Image analysis was carried out using Gwyddion and ImageJ. Statistical data are presented as mean ± standard error of the mean (SEM) unless stated otherwise.

The morphology of the GQDs was subjected to examination using transmission electron microscopy (TEM, FEI Talos F200S, New York, NY, USA) and atomic force microscopy (AFM, (Bruker Daltonics Inc. Multimode 8.0, Massachusetts, MA, USA). The Fourier transform infrared (FT−IR) spectra, UV−Vis absorption spectra, and FL spectra were obtained through utilization of FT−IR spectrometer (FT-IR 6800 JASCO, Marseille, France), Shimadzu UV−2450 spectrophotometer, and Hitachi 7000 fluorescence spectrophotometer, respectively. The hydrodynamic (HD) size and zeta potential of the samples were measured through the utilization of a nano ZS90 analyzer (Malvern Instruments Ltd., Worcestershire, UK), with the measurements being conducted at room temperature. The concentration of Gd³⁺ within the Gd(DTPA)−GQDs was confirmed through the application of inductively coupled plasma mass spectrometry (ICP-MS, Agilent 720 ES, Santa Clara, CA, USA).

The self-assembled structure H2 was investigated by atomic force microscopy (AFM). A solution of 1.00 × 10⁻⁴ M H1 was prepared in water. The solution of H2 were obtained by adding hydrochloride acid to the solution of H1. Then, the TEM samples of H2 was prepared by drop coating the solution on a Si substrate. AFM experiments were carried out on a Bruker Multi-Mode 8.0 instrument.

Atomic force microscopy was performed on bare mica and HOPG substrates. AFM maps were acquired by means of a Multimode VIII (Bruker), a NX10 (Park systems) and a nanowizard2 (JPK) system operating in tapping mode and equipped with a silicon tip (μmasch, 2 Nm⁻¹) with a nominal radius of 10 nm. Image flattening and single aggregate statistical analysis, such as nearest neighbor and cross-sectional dimension analysis, were performed by SPIP (Image Metrology) software.

The purified cellulose sample fractionations of D9 hypocotyls were performed as described previously (Li et al., 2017), with some minor modifications that hypocotyls were milled into fine powder under liquid nitrogen and then add 8% NaClO₂ (10 mL). The precipitated residue was treated with 4 M KOH for 1 h, washed with distilled water six times and resuspended in water for AFM scanning. The cellulose samples were suspended in ultrahigh‐purity water and placed on mica using a pipette. The mica was glued onto a metal disc (15 mm diameter) after removal of extra water under nitrogen and then placed on the piezo scanner of AFM (MultiMode VIII; Bruker, Santa Barbara, CA). AFM imaging was carried out in ScanAsyst‐Air mode using BrukerScanAsyst‐Air probes (tip radius, 2 nm and silicon nitride cantilever; spring constant, 0.4 N/m) with a slow scan rate of 1 Hz. All AFM images were 3rd‐flattened and analysed quantitatively using NanoScope Analysis software (Bruker). Three biological replications were performed each experiment, and 10 dots of each AFM image were randomly selected to measure the width (nm) × length (nm) by NanoScope Analysis software (Bruker). The average particle length/width of each image was calculated from the selected ten particles (n = 10).

The AFM images were taken in fluid cells using a Veeco Multimode VIII and Bruker ultra-sharp AFM cantilevers (MSNL-10). The amplitude set point was adjusted to 60–70% of the free amplitude value to decrease the damage to the protein superstructure.

AlproTox (WT, NB, or HB) solution (1 µg/ml) was deposited on the positively charged mica surface, pretreated by (3-aminopropyl) triethoxysilane vapour (APTES; Sigma-Aldrich, #919-30-2), and, scanned in peak force tapping mode in the fluid using a MultiMode VIII (Bruker, Santa Barbara, California, USA) by SNL-10C (Bruker) cantilever with a spring constant of 0,24 N/m and a tip radius of 2 nm. All parameters were optimised during imaging to avoid protein deformation. Data were processed using SPIP (Image Metrology, Lyngby, Denmark).