Bacterial cultures grown overnight were diluted 25-times in LB media and allowed to grow for 2 hours at 26°C to OD600 = 0.2. The growth conditions were then changed to T3SS-inducing conditions at 37°C. One milliliter was taken from the bacterial cultures every hour for analysis with atomic force microscopy. Each sample was centrifuged for 4 min at 1500 rpm, washed once with 2 mM MgCl2, and re-suspended in 50–200 μl of the same solution. Ten microliters of each sample was placed on freshly cleaved ruby red mica (Goodfellow Cambridge Ltd, Cambridge), incubated 5 min at room temperature, and blotted dry before being placed into a desiccator for a minimum of 2 hours. Images were collected by a Nanoscope V AFM (Bruker software) using ScanAsyst in air with ScanAsyst cantilevers at a scan rate of approximately 0.9–1 Hz. The final images were flattened and/or plane-fitted in both axes using Bruker software and presented in amplitude (error) mode.
Nanoscope 5 afm
Manufactured by Bruker
Sourced in United States
The Nanoscope V AFM is an atomic force microscope (AFM) system manufactured by Bruker. It is designed to provide high-resolution, three-dimensional imaging and characterization of surface topography and properties at the nanoscale. The Nanoscope V AFM utilizes a cantilever-based probe to scan the sample surface, allowing for the measurement of surface features with a high degree of precision and sensitivity.
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8 protocols using nanoscope 5 afm
1
Time-lapse Imaging of Bacterial T3SS
2
Characterization of Nanoparticle Systems
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The initial characterization study of FA-CFO-NPs
and FA-CFO-Dox-NPs was done using DLS to measure their mean particle
diameter and PDI. Further morphology of the NPs was determined by
using JEOL SEM and Bruker Nanoscope-V AFM.
and FA-CFO-Dox-NPs was done using DLS to measure their mean particle
diameter and PDI. Further morphology of the NPs was determined by
using JEOL SEM and Bruker Nanoscope-V AFM.
3
Atomic Force Microscopy of Self-Assembled Structures
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AFM-based
studies of the particles were carried out using tapping-mode
atomic force microscopy using Bruker Nanoscope-V AFM having optimum
scanning frequency of approximately 1 Hz and with several pixels of
approximately 512. A cantilever with a length of 196 μm was
used for the study; the spring constant was chosen to be 0.06 (N·m)−1 to determine the surface morphology and roughness
of the self-assembled structures. Samples prepared through self-assembly
were drop-cast on a silicon chip and air-dried before imaging.
studies of the particles were carried out using tapping-mode
atomic force microscopy using Bruker Nanoscope-V AFM having optimum
scanning frequency of approximately 1 Hz and with several pixels of
approximately 512. A cantilever with a length of 196 μm was
used for the study; the spring constant was chosen to be 0.06 (N·m)−1 to determine the surface morphology and roughness
of the self-assembled structures. Samples prepared through self-assembly
were drop-cast on a silicon chip and air-dried before imaging.
4
Characterizing Direct Tunneling in PVDF FTJs
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PFM loops versus voltage measurements (Figs 3a,b,e,f ) were performed at room temperature using an atomic force microscope (Bruker Multimode 8, Camarillo, CA, USA). PFM images (Fig. 1 ) were performed combining a lock-in amplifier (Zurich HF2LI) to track the contact resonance of the AFM tip with a multimode Nanoscope V AFM (Bruker). In both cases, commercial silicon tips coated with chromium/platinum (Budget Sensors) were used for PFM at typical contact resonance frequencies of 0.4–0.7 MHz.
I–V measurements were performed using a Keithley 6430 sub-femtoampere source meter with a remote preamplifier. Currents were collected 100 ms after the application of the external voltage. The bias voltage was applied to Au electrode both for PFM measurements and I–V measurements, while the other side (tip in PFM measurements and W electrode in I–V measurements) was grounded.
Temperature dependence of transport properties: Direct tunnelling can be experimentally distinguished from thermionic emission and Fowler–Nordheim tunnelling by its weak intrinsic temperature dependency of the resistance11 . I–V curves between −0.3 and 0.3 V for both ‘on' and ‘off' states of junction #1 were collected at 223, 240, 260 and 290 K. The current shows low variations with temperature (Supplementary Fig. 3 ), which strongly suggests that direct tunnelling dominates the current flow in PVDF FTJs.
I–V measurements were performed using a Keithley 6430 sub-femtoampere source meter with a remote preamplifier. Currents were collected 100 ms after the application of the external voltage. The bias voltage was applied to Au electrode both for PFM measurements and I–V measurements, while the other side (tip in PFM measurements and W electrode in I–V measurements) was grounded.
Temperature dependence of transport properties: Direct tunnelling can be experimentally distinguished from thermionic emission and Fowler–Nordheim tunnelling by its weak intrinsic temperature dependency of the resistance11 . I–V curves between −0.3 and 0.3 V for both ‘on' and ‘off' states of junction #1 were collected at 223, 240, 260 and 290 K. The current shows low variations with temperature (
5
Fungal Cell Surface Nanomechanics Analysis
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The surface of both the control and AAF-treated fungal cells was measured using NanoScope V AFM (atomic force microscope) in the Peak-Force Quantitative Nanomechanical Mapping Mode (Bruker, Vecco Instruments Inc., Billerica, MA, USA) and NanoScope 8.15 software. The nominal spring constant of the RTESPA probe (Bruker, Billerica, MA, USA) (silicone tip on the nitride lever) was 40 N/m.
6
Algal Cell Surface Characterization by AFM
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Algal cells prepared as for the SEM analysis (as above) in 100% acetone were placed on a mica disc and analyzed using atomic force microscopy (AFM). The cell surface was characterized using a NanoScope V AFM (Bruker, Vecco Instruments Inc., Billerica, MA, USA) in the Peak-Force Quantitative Nanomechanical Mapping Mode and NanoScope 8.15 software. The nominal spring rate of the RTESPA probe (Bruker, Billerica, MA, USA) (silicone tip on nitride lever) was 40 N/m.
7
Atomic Force Microscopy of CH4/Ibu on Mica
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As a support for the CH4/Ibu sample was used a freshly cleaved mica surface type Grade V-4 Muscovite (Structure Probe Inc./SPI Supplies, West Chester, PA). The squared mica sheet with size 10 mm was glued to the metal pad. The opalescent suspension of solid particles of CH4/Ibu, before applying it to the mica surface, was placed in an ultrasonic probe sonicator for 10 min until its partial clearing and 100 μL of diluted suspension of CH4/Ibu was spread on the mica surface. The tested sample was left for 10 min, and then was carefully dried with a mild flow of nitrogen gas.
AFM studies were carried out using NanoScopeV AFM (Bruker Inc.) at air in tapping mode and room temperature. A silicon cantilevers were used (Tap 300Al-G, Budget Sensors, Innovative solutions Ltd, Bulgaria) with reflective aluminum coating with thickness of 30 nm. The characteristics of the cantilevers as indicated by the manufacturer are as follows: spring constant of 1.5 to 15 N/m, resonance frequency of 150 ±75 kHz and radius of curvature of the tip less than 10 nm. The scanning rate was set at 1 Hz. All images were taken at resolution of 512×512 pixels in JPEG format and further were processed by means of Nanoscope software. Images from three independent locations of the samples were taken for reproducibility purposes.
AFM studies were carried out using NanoScopeV AFM (Bruker Inc.) at air in tapping mode and room temperature. A silicon cantilevers were used (Tap 300Al-G, Budget Sensors, Innovative solutions Ltd, Bulgaria) with reflective aluminum coating with thickness of 30 nm. The characteristics of the cantilevers as indicated by the manufacturer are as follows: spring constant of 1.5 to 15 N/m, resonance frequency of 150 ±75 kHz and radius of curvature of the tip less than 10 nm. The scanning rate was set at 1 Hz. All images were taken at resolution of 512×512 pixels in JPEG format and further were processed by means of Nanoscope software. Images from three independent locations of the samples were taken for reproducibility purposes.
8
Electrical Surface Characterization via EFM
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Electrical measurements on the sample surface were also performed using the Nanoscope V AFM (Bruker). EFM images were taken using a double pass approach [36] [37] [38] . In this work, EFM experiments were carried out using Pt-Ir covered Si probes (SCM-PIT, Bruker, k = 4 N/m). Lift height was kept constant at 80 nm above each point on the surface, and the applied tip voltage was +5 V.
Lift height was chosen in order to avoid effect of the topography.
Lift height was chosen in order to avoid effect of the topography.
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