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10 protocols using nanoscope 9

1

Optimized AFM Protocol for Cell Mechanics

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The measurement method was slightly modified from our previous publication (Iwashita et al., 2014 (link)) to optimize for fixed samples. The measurements were carried out using AFM (Bioscope Resolve, NanoScope 9.4, Bruker), which was mounted on an inverted microscope (Nikon, ECLIPSE Ti2). A tipless silicon cantilever with a 20-μm borosilicate bead (Novascan) was used. The spring constant of the cantilever was calibrated using the thermal noise method in air. We chose cantilevers with the same spring constant (nominal value: 0.03 N/m; actual value: 0.07 N/m) and used them for acute and fixed slices individually to avoid cross-contamination of the remaining fixative in acute condition. The applied force was 10 nN. The measurement was done under physiological conditions for the acute slices (37°C) and at room temperature (25°C) for fixed slices. The force curves were acquired using the contact mode. Bright field images were acquired by a CMOS camera (Hamamatsu, ORCA-Flash4.0, C13440-20CU) to determine the measured region. The obtained force curves were analyzed to calculate the stiffness fit with the Hertzian model (spherical) using NanoScope Analysis 1.9 software (Bruker). Supplementary Table 3 describes the parameters for measurement.
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2

Atomic Force Microscopy of Microgels

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Deposited, dried microgels were imaged using a Dimension Icon atomic force microscope with closed loop (Veeco Instruments Inc., USA, Software: Nanoscope 9.4, Bruker Co., USA) in tapping mode. The probes were OTESPA tips with a resonance frequency of 300 kHz, a nominal spring constant of 26 Nm−1 of the cantilever and a nominal tip radius of <7 nm (Opus by Micromasch, Germany).
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3

AFM Imaging for Cytoskeleton Analysis

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Initial AFM imaging was carried out by using a MultiMode AFM with Nanoscope V electronics (Bruker AXS SAS, Santa Barbara, CA) controlled with Nanoscope 9.2 software (Build R2Sr1.130547). Scans were made in ac mode using a silicon probe (Olympus Co. AC240) or in PeakForce Tapping mode using a Super Sharp Silicon tip on a silicon nitride lever (Bruker Co. SAA-HPI-SS).
The large amount of images necessary to train and test the CNN model required high image throughput. This was achieved with a fast-scan AFM system, using photothermal off-resonance tapping and small cantilevers, which fits on the base of the commercial MultiMode AFM system. The fast scanning AFM head and electronics were designed by Georg E. Fantner’s group, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland98 (link), images were collected with custom software from the group of Georg Fantner, EPFL, (IbniAFMController-BetaVersionv2.0.16-20190117 written under Labview environment). Scans were made with a silicon tip on a silicon nitride cantilever- (Bruker FASTSCAN-B). All cytoskeleton images were analyzed using Gwyddion 2.55 software99 .
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4

Atomic Force Microscopy Nanoindentation Protocol

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The samples were prepared by incubation on mica as for the imaging experiments above. Images and mechanical properties were obtained simultaneously using “peak force QNM” on a Multimode AFM (Bruker), under ambient conditions. Tap525A probes, with a nominal spring constant (k) of 200 N/m or RTESP-300-30 (Bruker) with a calibrated spring constant of 47.1 N/m probes were used. The effective tip radius was calibrated on a sample of HOPG with a modulus of 18 GPa or on PolyStyrene (PS) with a modulus of 2.7 GPa. Deformation depths were kept to about 1 nm. The Bruker software (Nanoscope 9.2) was used to compute the elastic modulus maps using the DMT model to fit the force vs. deformation curves.
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5

Nanomechanical Characterization of Biological Fibrils

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Imaging and nanomechanical properties measurements were conducted using PeakForce QNM on a Multimode AFM (Bruker). AC160 probes (Olympus) with a spring constant of 40 N/m were used. The effective tip radius was calibrated on a sample of HOPG with a modulus of 18 GPa, deformation depths were kept to about 1 nm. The Bruker software (Nanoscope 9.2) was used to compute the elastic modulus maps using the DMT model for fitting the force vs. deformation curves. Gwyddion open-source software was used for the image analysis. All averaged modulus values were calculated from the center of each fibril or aggregate.
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6

Hydrogel Stiffness Measurement by AFM

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The stiffness of hydrogel was estimated using Bioscope Catalyst Atomic Force Microscope (Bruker) with a 2.5-μm radius, sphere-tipped AFM tip (Novascan). The spring constant of the AFM tip was determined at ~120 pN/nm by thermal fluctuation method (46 ). Hydrogel samples were submersed in PBS and indented at low indentation rate (0.5 Hz) with a trigger force of 5 nN. Twenty-five force curves were performed over a 100 μm by 100 μm area (grid of 5 × 5 points separated by 20-μm distance) using MIRO 2.0 (NanoScope 9.1; Bruker). The elastic modulus (Young’s modulus) E was then extracted from each force curve by the fitting (NanoScope Analysis software, v1.8, Bruker) with the Hertz contact model for spherical indenter and following the relationship (Eq. 1) E=34 (1ν2)R1/2δ3/2F with R as the tip radius, ν the Poisson’s ratio defined at 0.45 for hydrated hydrogels, and δ as the sample indentation. In total, two areas per hydrogel sample were analyzed, and two samples were prepared per hydrogel stiffness value (n = 3).
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7

Atomic Force Microscopy and Mass Spectrometry

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AFM analysis was performed on a Bruker Dimension Icon using NanoScope 9.1 (Karlsruhe, Germany) for measurements and NanoScope Analysis 1.5 for image processing. We measured in ScanAsyst air mode using a ScanAsyst air tip with a spring constant of ~0.4 N/m and a resonant frequency of 70 Hz.3.2.4. NMR and ESI MS NMR spectra were recorded on a Bruker Avance 300 MHz Spektrometer (Ettlingen, Germany). Mass spectra were recorded on FlexarTM SQ 300 MS Detector (PerkinElmer, Rodgau, Germany).
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8

Nanomechanical Characterization of Multilayered Films

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The multilayered films were imaged using a dimension Icon microscope controlled by NanoScope 9.1 (Bruker, France), operating in peak force tapping (ScanAsyst) mode with quantitative nanomechanical analysis, following established protocols.[51 (link), 52 ] AFM silicon nitride cantilevers (ScanAsyst-Air, Bruker) with a spring constant of 0.5 N/m, a frequency of 70 kHz, and a radius of the tip of 5 nm were used. The peak-force set-point was adjusted to 3.0 μN and the Poisson’s ratio was assumed to be equal to 0.3 for the entire 512×512 pixels (2×2 μm2) scan of the sample. The line scan rate was 0.4 Hz. The root mean squared roughness (RRMS), average height value (Hav), and average elastic modulus (Eav) were calculated for each formulation in the wet state. Image analysis was performed using Gwyddion and Nanoscope Analysis 1.5. At least three measurements were performed indifferent specimens.
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9

Comprehensive Characterization of Cellulose Nanofibrils

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For the viscosity
measurements, a DV2TLV viscometer (Brookfield) was used along with
Rheocalc software. Each CNF sample (0.4% CNF suspension) was evaluated
in three parallels, and the viscosity of each parallel was measured
from 0.1 to 100 rpm. For the nanostructures, 0.01% nanocellulose suspensions
of each sample (25 μL) was added to clean the mica surface,
left to dry in air, and then imaged with a Dimension ICON atomic force
microscope (AFM) using NanoScope 9.4 Software (Bruker). Surface roughness
(Ra) was obtained from images (10 μm
× 10 μm) using NanoScope Analysis software (version 1.9).
For the quantification of residual fiber content, a Fiber Tester
912 Plus (ABB AB/Lorentzen Wettre) was used. CNF suspensions were
pumped through a flow cell where the fibers were photographed with
a resolution of 4 μm. The images were further used to analyze
the mean length and width of the fibers. To evaluate the microstructure
of all cellulosic materials, tissue culture plates (24-well clear
flat bottom polystyrene; NUNC, Denmark) were covered with cellulosic
suspensions and stored at 37 °C for 24 h to dry. Films were analyzed
using an optical microscope (Nikon Eclipse 80i, Tokyo, Japan) after
staining with crystal violet.
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10

Adsorption of Emulsion Droplets on Mica Substrate

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A 5 µL volume of the freshly prepared NFPh emulsion, prepared as described in Section 4.1, was dispensed onto freshly cleaved mica. Mica surface (0.15 mm thick, sized 15 × 15 mm, TipsNano, Zelenograd, Russia) was used as a substrate for non-covalent adsorption.
The emulsion droplet was incubated on the mica substrate surface for 10 min. Then, the substrate was washed with 1 mL of deionized water, which was obtained using a Simplicity UV system (Millipore, Molsheim, France). The washed substrate was dried in air and subjected to AFM scanning.
The AFM images of nanosized particles were obtained with a Dimension atomic force microscope equipped with an Icon scanner (Bruker, Billerica, MA, USA). The instrument is a part of the Avogadro unique research facility (http://avo.ibmc.msk.ru/ (accessed on 1 October 2023)). Scanning was carried out in the tapping mode. A short cantilever holder was used; the measurements were conducted in air. The images were recorded using the NanoScope 9.4 software (Bruker, Billerica, MA, USA). AFM images were processed using the standard NanoScope Analysis 2.0 software (Bruker, Billerica, MA, USA), Gwyddion 2.62, and Femtoscan Online software 4.8 (LLC Scientific and Production Enterprise “Center for Advanced Technologies”, Moscow, Russia; www.nanoscopy.net/en/Femtoscan-V.shtm (accessed on 6 May 2021)).
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