A 4ul aliquot of dispersion of gold nanoparticles was placed on a quantifoil grid (1.2/1.3, EMS) and then a filter paper was applied to remove excess dispersion. For MUS-OT, no salt was added to the sample. For MUA-OT, a salt concentration of 20 mM NaOH was added to the sample. The thin film of dispersion was vitrified by liquid ethane in a homemade plunge freeze device. Vitrification rate was of the order of 107 K s−1 (ref. 64 (link)) so that the population of aggregates does not vary during the freezing process. The vitrified grid was loaded to a cryogenic holder Gatan 626 (Gatan, USA) and transferred to a transmission electron microscope F20 (Thermofisher, USA). Images were recorded in low dose mode at magnification of 50 000× at 4096 × 4096 pixels. Tilt series were acquired from −60° to 60° at an incremental angle of 2° by Tomography 4.0 (Thermofisher, USA). The tilt series images were binned by 2 and then aligned by Inspect3D (Thermofisher, USA). The reconstruction was carried out in Inspect3D (Thermofisher, USA) using SIRT algorithm, at the final pixel size of 0.41 nm. The identification of locations of nanoparticles in the tomograms was carried out in Imaris (Oxford Instruments, UK) and the 3D centroid positions were exported to calculate radial distribution functions.
Inspect3d
Manufactured by Thermo Fisher Scientific
Sourced in United States
Inspect3D is a high-performance 3D imaging and measurement system designed for a wide range of industrial applications. It provides precise and accurate 3D data capture, analysis, and reporting capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Gatan 626, by Ametek (1 mentions)
Imaris, by Oxford Instruments (1 mentions)
Xplore3d, by Thermo Fisher Scientific (1 mentions)
Tecnai spirit biotwin tem, by Thermo Fisher Scientific (1 mentions)
Vitrobot, by Thermo Fisher Scientific (1 mentions)
Talos arctica, by Thermo Fisher Scientific (1 mentions)
Tecnai f20 feg tem, by Thermo Fisher Scientific (1 mentions)
5 protocols using inspect3d
1
3D Imaging of Gold Nanoparticle Aggregates
2
Electron Tomography Reconstruction and Analysis
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All of the tilt series were aligned by cross-correlation, followed
by manual adjustment of the tilt axis shift and reconstructed by SIRT
with 25 iterations in Inspect 3D (Thermo Fisher Scientific, MA, USA).
BF-STEM and EDX tilt series were aligned based on the HAADF-STEM tilt
series. The reconstructed electron tomograms were visualized and analyzed
with Dragonfly (Object Research Systems, QC, Canada), as described
below.
by manual adjustment of the tilt axis shift and reconstructed by SIRT
with 25 iterations in Inspect 3D (Thermo Fisher Scientific, MA, USA).
BF-STEM and EDX tilt series were aligned based on the HAADF-STEM tilt
series. The reconstructed electron tomograms were visualized and analyzed
with Dragonfly (Object Research Systems, QC, Canada), as described
below.
3
Tilt Series Tomography of Skin Biopsies
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Two hundred nm sections of embedded skin biopsy specimens were collected on glow discharged carbon grids, and 10 nm colloidal gold fiducial markers were applied. Using a linear tilt scheme, single axis tilt series were collected on a Tecnai BioTwin Spirit TEM (FEI) operated at 120 kV. Images captured over a tilt range of +/- 68° (1° increments) at 1 μm defocus were recorded on a 2048 x 2048 pixel UltraScan 1000 Gatan CCD camera using the automated tomography acquisition software (Xplore 3D, FEI). Resulting images had a binning factor of 1 and pixel size of 2.1 nm. The tilt series were aligned using either Inspect 3D (FEI) or IMOD software package (version 4.2.5) and SIRT reconstructions of 35 iterations were performed. Movies were generated through the volume ortho-slices and volren models created from unfiltered tomograms with inverted contrast by thresholding colors based on pixel intensity within the volumes using the Amira Visualization Package (version 5.3.0, Visage Imaging, Carlsbad, CA).
4
Cryo-Electron Tomography of MalaEx
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Vitrification of MalaEx isolated from the RPMI cultures was performed with a Vitrobot (FEI Company; OR, USA). The samples were blotted for 3 sec and plunge frozen in liquid ethane. Tilt series were collected with SerialEM46 (link) at 3° increments over a range of ±60°, with a defocus of 4 to 6 µm. Images were recorded on a 200 kV Talos Arctica (FEI Company) using a Falcon 2 direct electron detector (FEI) at a nominal magnification of 22 000× (corresponding to a pixel size of 6.61 Å). Fiducial-free alignment of tilt images and tomogram reconstructions was carried out in IMOD using the patch-track procedure47 (link). Four tomograms were reconstructed and non-linear anisotropic diffusion (NAD) filtered48 (link). Two tomogram were collected with the Volta phase plate using the Tomo software (FEI company) and reconstructed with Inspect3D (FEI company). Movies were generated using Fiji-ImageJ (imagej.net/Fiji open source image processing package).
5
Electron Tomography of Gold Nanoparticles
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Electron tomography tilt series data was acquired using an FEI Tecnai F20 FEG-TEM operated at 200 kV. Gold nanoparticles were prepared by a modified seed-mediated growth process as reported by Bandhopadhyay et al. (2017) . The sample was drop cast onto 200 mesh copper TEM grids coated with a continuous carbon film. A tilt series of HAADF STEM images was acquired from -68° to +64° in 2° increments. The tilt series image stack was first aligned using cross-correlation methods followed by alignment of the tilt axis, performed using Inspect3D (FEI).
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