To gain information about the surface topography AFM surface roughness measurements were used. The AFM measurements were conducted with a NanoWizard™ UltraSpeed 2 (JPK-Bruker, Berlin, Germany). The data were recorded using a Tap300Al-G cantilever from BudgetSensors® (Innovative Solution Bulgaria Ltd., Sofia, Bulgaria) operating in tapping mode. The data were evaluated with the open-source software Gwyddion [18 (link)].
Tap300al g cantilever
Manufactured by Budget Sensors
Sourced in Bulgaria
The Tap300Al-G cantilevers are a type of lab equipment designed for use in atomic force microscopy (AFM) applications. They feature an aluminum coating and have a resonant frequency range of 200-400 kHz. The core function of these cantilevers is to facilitate the detection of surface topography and material properties during AFM measurements.
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Lab products found in correlation
Escalab 250, by Thermo Fisher Scientific (3 mentions)
Multimode 8 afm, by Bruker (2 mentions)
Multimode afm, by Digital Instruments (1 mentions)
Axiovert 200m epifluorescence microscope, by Zeiss (1 mentions)
Nicolet 6700 spectrometer, by Thermo Fisher Scientific (1 mentions)
Hr800 spectrometer, by Horiba (1 mentions)
Flexafm atomic force microscope, by Nanosurf (1 mentions)
C3000 controller, by Nanosurf (1 mentions)
Axio observer, by Zeiss (1 mentions)
Mfp 3d afm, by Oxford Instruments (1 mentions)
Dynapro99, by Wyatt Technology (1 mentions)
8 protocols using tap300al g cantilever
1
AFM Surface Roughness Characterization
2
Tapping Mode AFM Measurements
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The AFM measurements were conducted with a NanoWizard™ Ultra Speed 2 (JPK-Bruker, Berlin, Germany). The data were recorded using a Tap300Al-G cantilever from Budget Sensors® (Innovative Solution Bulgaria Ltd., Sofia, Bulgaria) operating in tapping mode. The data were evaluated with the software provided by the AFM manufacturer.
3
Graphene Oxide Synthesis and Characterization
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Graphene Oxide Synthesis and Characterization. GO was produced by chemical oxidation of graphite by KMnO 4 in a mixture of H 2 SO 4 and H 3 PO 4 , as previously described. 33 Spectroscopic characterization was realized on dry GO powders. Raman spectroscopy was performed on a Horiba Jobin Yvon HR-800 spectrometer with a 532 nm excitation. Fourier-Transformed Infrared (FTIR) spectra were collected using a Thermo Nicolet 6700 spectrometer.
X-ray photoelectron spectroscopy (XPS) was performed on a ThermoScientific ESCALAB 250 with a monochromatized AI X-ray source. For microscopy analysis, GO sheets were drop-casted on a silicon wafer. Atomic Force Microscopy analysis was performed in tapping mode with a Bruker Multimode AFM (Digital Instruments, Plainview, NY) equipped with a Tap300Al-G cantilever (BudgetSensors, Sofia, Bulgaria). SEM analyses were done on a Hitachi SU-70 microscope (Hitachi High Technologies America, Inc., Clarksburg, MD). The antimicrobial activity of GO was verified by measuring the cell viability of P. aeruginosa cells deposited on a pure GO layer. Cell viability was measured after 1 h by staining the cells with SYTO 9 and propidium iodide (PI) and quantifying live and dead cells with an Axiovert 200M epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY). Further information on GO synthesis and characterization is given in the Supporting Information (SI).
X-ray photoelectron spectroscopy (XPS) was performed on a ThermoScientific ESCALAB 250 with a monochromatized AI X-ray source. For microscopy analysis, GO sheets were drop-casted on a silicon wafer. Atomic Force Microscopy analysis was performed in tapping mode with a Bruker Multimode AFM (Digital Instruments, Plainview, NY) equipped with a Tap300Al-G cantilever (BudgetSensors, Sofia, Bulgaria). SEM analyses were done on a Hitachi SU-70 microscope (Hitachi High Technologies America, Inc., Clarksburg, MD). The antimicrobial activity of GO was verified by measuring the cell viability of P. aeruginosa cells deposited on a pure GO layer. Cell viability was measured after 1 h by staining the cells with SYTO 9 and propidium iodide (PI) and quantifying live and dead cells with an Axiovert 200M epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY). Further information on GO synthesis and characterization is given in the Supporting Information (SI).
4
Atomic Force Microscopy of rAAV2 Variants
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AFM measurements of rAAV2 wt and rAAV2_587_bla were performed on a Multimode 8 AFM (Bruker, Karlsruhe, Germany) with Tap300Al-G cantilevers (BudgetSensors, Sofia, Bulgaria) in tapping mode in air. 2 µL of sample in PBS were spotted onto freshly cleaved mica and incubated for one minute. The mica was then briefly rinsed with water and dried under a gentle nitrogen flow. Data analysis was performed with Gwyddion 2.48. Obtained images were treated with offset and plane correction algorithms and the size of visualized particles was measured at half maximum particle height. Statistical analysis of size measurements was performed using Excel 2016.
5
Multimodal Imaging of Transfected Cells
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After seeding the cells onto glass bottom dishes, in-vitro transfection was performed using 5 nM (low) and 100 nM (high) final nucleic acid concentrations of N-TER peptide–FluoNTC complexes followed by formaldehyde fixation of 3T3-L1 cells 4-hr post-transfection. The samples were investigated by using a combinatorial set up including AFM, BF and fluorescence imaging. Mounting the atomic force microscope on top of a Zeiss Axio Observer (Göttingen, Germany), this instrumental set up combines the three different microscopic techniques enabling simultaneous investigations at the very same sample area. The AFM investigations were performed with a FlexAFM atomic force microscope, equipped with a C3000 controller (Nanosurf, Switzerland). Height images were recorded in tapping mode utilizing Tap300 Al-G cantilevers (Budgetsensors, Sofia, Bulgaria). All recorded AFM data were processed and examined with the software package Gwyddion.44 (link) For fluorescence microscopy, filter settings were chosen according to the absorption/emission spectra (557 nm/570 nm) of the fluorescence marker Dy547. Combined images of all techniques were generated with the software GIMP.
6
Nanoscale Topography and Phase Analysis
7
Characterization of Recombinant Viral Particles
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Dynamic light scattering (DLS) was performed on a DynaPro99 (Wyatt Technology) instrument. Samples were filtered through 0.22 µm PVDF syringe filters (Millipore) and centrifuged at 18,000 × g for 30 min before measurement. Measurements for each sample were averages of at least 20 acquisitions.
Atomic Force Microscopy (AFM) measurements of rAAV2 and VLPs were carried out using a Multimode 8 AFM (Bruker) with Tap300Al-G cantilevers (BudgetSensors) in tapping mode in air. Samples after filtration and centrifugation were spotted onto freshly cleaved mica and incubated for one minute. The mica was then briefly rinsed with water and dried under a gentle nitrogen flow. The analysis of the AFM images was performed with Gwyddion 2.49 software. The diameter of visualized particles was measured at half maximum particle height. The rAAV used as control was provided by K. Teschner and was produced using a three plasmid system in HEK-293 cells and purified by affinity chromatography.
Atomic Force Microscopy (AFM) measurements of rAAV2 and VLPs were carried out using a Multimode 8 AFM (Bruker) with Tap300Al-G cantilevers (BudgetSensors) in tapping mode in air. Samples after filtration and centrifugation were spotted onto freshly cleaved mica and incubated for one minute. The mica was then briefly rinsed with water and dried under a gentle nitrogen flow. The analysis of the AFM images was performed with Gwyddion 2.49 software. The diameter of visualized particles was measured at half maximum particle height. The rAAV used as control was provided by K. Teschner and was produced using a three plasmid system in HEK-293 cells and purified by affinity chromatography.
8
Atomic Force Microscopy Protocol
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Studies using atomic force microscopy (AFM) were conducted with a non-contact PSIA XE-70 microscope fitted with Budget Sensors silicon probes (TAP-300AL-G cantilevers, 300 kHz resonance frequency; spring constant of 40 Nm−1). We used Gwyddion® software to correct sample inclination and distortions caused by the z-scanning stage [34 (link)].
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