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8 protocols using picoview software

1

Measuring Cellular Biomechanics with AFM

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Cells were detected by a contact mode PicoPlus AFM controlled by Picoview software (Agilent Technologies). Biomechanical properties were calculated from in situ force–distance curve measurements in medium at room temperature. The radius of silicon nitride tips was 20 nm. Its spring constant was calibrated to 0.10–0.11 N m−1 by Thermo K Calibration (Agilent Technologies) and its corresponding deflection sensitivities were 45–50 nm V−1. More than 10 cells were detected, collecting at least 15 force curves on the central area of different cells to avoid spurious detections.25 (link),26 (link) Scanning Probe Image Processor (SPIP) software (Image Metrology) was used to calculate Young’s modulus and adhesion force by fitting the Sneddon variation of Hertz model.27 –29 (link) The half cone-opening angle of tip was 36°, and cellular Poisson’s ratio was 0.5. The detection was accomplished within 2 hours (h) to approximate physiological conditions.
Ecell = 4FZ)(1 − ηcell2)/3(ΔZ1.5)tan θ, where Ecell: Young’s modulus; F: loading force; ηcell: Poisson ratio; ΔZ: indentation; θ: tip half cone opening angle.
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2

Atomic Force Microscopy of Chondrocyte-Derived Extracellular Vesicles

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MVs were isolated from differentiating chondrocytes by differential centrifugation as described earlier.(9 (link)) A drop (5 µL) of each MV solution in Tris-buffered-saline was spotted on freshly cleaved mica substrates (Ted Pella, Redding, CA) and allowed to stand for 5 min. Next, 5 µL of glutaraldehyde solution (8% in H2O, Sigma-Aldrich, St. Louis, MO) was dropped onto the samples. The substrates were stored inside a desiccators at room temperature for 24 h. AFM images of dried samples were recorded in air by means of an 5500 atomic force microscope (Agilent Technologies, Santa Clara, CA) equipped with an open-loop probe working in non-contact (AAC) mode. Silicon-nitride cantilevers having a nominal resonance frequency of ~190 kHz (NanosensorsTM, Neuchatel, Switzerland) were used. Tridimensional AFM images were generated by PicoView software (Agilent Technologies). AFM images were used to gather information about the morphology, height, volume and number of MVs in each sample. AFM phase images were also recorded on samples prepared without the use of glutaraldehyde.
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3

Atomic Force Microscopy Imaging of Lysozyme Fibrils

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For the AFM imaging
study, the solutions
of lyz fibrils were diluted to 100-fold with water. Then, around 5
μL of this sample solution was adsorbed onto a freshly cleaved
muscovite ruby mica sheet (ASTM VI grade Ruby Mica from Micafab, Chennai,
India). Thereafter, the mica sheet was dried for 30 min in vacuum
in an inert atmosphere. The complexes were incubated for 15 min prior
to adsorption onto the mica sheet. AFM was performed in the AAC mode
on PicoPlus 5500 ILM AFM (Agilent Technologies, USA), which was attached
with a piezo-scanner of a maximum range of 9 μm. Here, microfabricated
silicon cantilevers of NANOSENSORS (USA) were used. The resonance
frequency of the cantilever oscillation was 146–236 kHz, whereas
the force constant was 21–98 N/m. The rate of the scan speed
was 0.5 lines/s while taking the images (256 by 256 pixels). All the
images were processed by flattening using PicoView software (Agilent
Technologies, 1.1 version), whereas their manipulation was conducted
by Pico Image Advanced version software.
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4

AFM Characterization of Cells and Collagen I Gel

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For AFM characterization of cells and collagen I gel (see Supplement for gel formation methodology), a Molecular Imaging-Agilent PicoPlus AFM system was used. Imaging of fixed cells was performed in contact mode in air and under phosphate buffered saline (PBS) with silicon and V-shaped silicon nitride probes (SICON-Applied Nanostructures and PNP-TR-Nanoword). Force spectroscopy on live cells was performed with V-shaped soft silicon nitride probes (MLCT, Bruker) and the collected force curves were analyzed by AtomicJ (29 (link)) so as to calculate the sample’s Young’s modulus using the Hertz model. Collagen I gels were characterized with V-shaped PNP-TR probes in contact mode. The AFM image processing from all the samples was performed by using the PicoView software (Agilent) and the freeware scanning probe microscopy software WSxM 5.0 dev.2.1 (30 ). A detail representation of the AFM methods is presented in the Supplement.
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5

Atomic Force Microscopy of Polymer-DNA Complexes

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AFM imaging was done using an
Agilent 5500 microscope without pretreatment of the sample. To take
the images of polymer–DNA complexes, the contact mode in air
was conducted on mica. The microfabricated Si-type NCH cantilevers
had a nominal spring constant of 0.2 N/m and a nominal resonance frequency
of 13 kHz. The scan rate employed was below 2 Hz to obtain good tracking
of the surface morphology. The polymer–DNA complexes were prepared
at Z+/– = 1 and kept at two different
temperatures 25 and 40 °C for 30 min, and then, a drop of the
sample solution was allowed to settle on the mica for 5 min. The samples
were air-dried overnight at respective temperatures. The images were
further autoflattened and analyzed using Agilent PicoView software.
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6

Atomic Force Microscopy of Plastoglobules

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AFM measurements were performed with an Agilent 5500 (Agilent Technologies Inc., Santa Clara, CA, USA) microscope, working in contact and tapping modes, under the control of PicoView software (Agilent Technologies Inc., Santa Clara, CA, USA). Samples were imaged in a liquid cell, and SNL probes (Bruker Corp., Billerica, MA, USA) with nominal k = 0.35 N/m were used. For elasticity determination, the k constant of each probe was measured with a built-in Agilent Thermal-K setup. Plastoglobule samples were immobilised on freshly cleaved mica, with poly-L-lysine used as stabilising matrix.
Images were recorded at 1 ln/s and resolution 512 × 512, with minimal possible force applied. Surface probing for elasticity determination was done with a built-in plugin for PicoView software, recording and analysing force-distance curves. Usually, a resolution of 32 × 32 was used for probing a chosen region of approximately 2 μm × 2 μm. Images were processed with Gwyddion 2.49 software [79 (link),80 (link)].
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7

Atomic Force Microscopy of Membrane Vesicles

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Five microliters of MV solution was dropped onto a freshly cleaved mica substrate (Ted Pella, Redding, CA) and allowed to stand for a couple of minutes. Next, the substrate was rinsed with ddH2O and dried at room temperature overnight. Samples were imaged by non-contact (AAC) mode in air using a 5500 AFM (Agilent Technologies). Silicon-nitride cantilevers with a nominal resonance frequency of ~190 kHz (NanosensorsTM, Neuchatel, Switzerland) were employed. Topography, amplitude and phase images were recorded for each scanned field and three-dimensional AFM images were generated by PicoView software (Agilent Technologies).
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8

Characterizing Thin Film Morphology and Composition

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AFM
amplitude micrographs were obtained in tapping mode using a Pico Plus
AFM instrument (Molecular Imaging) with aluminum-coated silicon tips
(BudgetSensors) and a spring constant of 40 N m–1. A sample area of 3 μm by 3 μm was scanned at a rate
of 1 Hz while collecting data in topographic, phase, and amplitude
modes. Morphological changes are captured in micrographs using PicoView
software (Agilent) and postprocessed using Gwyddion.30 (link)FTIR imaging was performed with a Nicolet iN10 infrared
microscope (Thermo Scientific) after mounting the optical windows
on a motorized stage for scanning the infrared map in an XY pattern. OMNIC Picta software (Thermo Scientific) was utilized for
FTIR microscopy and spectral mapping. Individual spectra corresponding
to an average of 64 scans were collected over the range of 800–4000
cm–1 with 4 cm–1 resolution. All
samples were background-subtracted using an empty optical window.
Control experiments ensured that films exposed to humid air in the
absence of O3(g) correspond to the spectral features of
catechol despite any loss by sublimation, which was carefully monitored
to remain below 5%. Data processing to obtain the CD line (or corrected
peak heights after local baseline correction) was performed10 (link) to collect kinetic data from the average of
duplicate experiments with error bars corresponding to one standard
deviation.
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