To characterise the particle size distribution and morphology of the iron oxide nanoparticles, very small droplets of the individual FeOxNP dispersions and the mixture were pipetted onto TEM grids and the excess moisture was allowed to dry. To measure the size of the primary particles, TEM imaging of samples was carried out using a FEI Tecnai F20 Transmission Electron Microscope working on high tension at 200 kV, with an extraction voltage of 4450 eV. Bright-field particle imaging in TEM mode was carried out using the Gatan Digital Micrograph attached to the instrument. For identifying likely particles, the instrument scanning TEM (STEM) mode was used in conjunction with high angle annular dark field (HAADF) detection. The Gatan Digital Micrograph software was used to measure the particle diameters of 40 NPs of each iron oxide. Additional high-resolution TEM images were taken with a JEOL 3000F Transmission Electron Microscope (JEOL Inc., Tokyo, Japan).
Gatan digital micrograph
Manufactured by Ametek
Sourced in United States
Gatan Digital Micrograph is a software suite used for acquiring, processing, and analyzing digital images from various types of microscopes, including electron microscopes. It provides a comprehensive set of tools for image manipulation, measurement, and analysis, enabling researchers and scientists to extract valuable information from their microscopic samples.
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7 protocols using gatan digital micrograph
1
Characterization of Iron Oxide Nanoparticles
2
Electron Microscope Image Analysis
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Electron microscope images were obtained with a LEO 910 Electron Microscope. Images were analyzed with Gatan Digital Micrograph provided by Gatan, Inc. (Pleasanton, CA, USA).
3
Transmission Electron Microscopy Sample Preparation
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Cells were briefly washed in PBS prior to fixation in 2.5 % glutaraldehyde (1 h or O/N in 4 °C). The dishes containing the cells were rinsed in 0.15 M Na-caccodylate and incubated for 1 h in 1 % osmium tetroxide in Na-caccodylate at RT followed by Na-caccodylate rinse. Dehydration was performed with 70 % ethanol for 30 min, 95 % ethanol for 30 min and 99.7 % ethanol for 1 h. A thin layer of newly made plastic (Agar 100 resin kit, Agar Scientific Ltd) was added for 1 h to permit evaporation of the alcohol. The plastic was poured off and a second plastic layer was added and left O/N in a desiccator. Next, plastic was heated up in an oven to enable its removal and a thicker, newly made plastic layer was added. The dishes were incubated in desiccator for 1–4 h before polymerization in oven (60 °C) for 48 h. Cells were sectioned using Leica ultracut UTC ultrotome (Rowaco AB) and studied in a Tecnai G2 transmission electron microscope (FEI Company). ORIUS SC200 CCD was used as camera and Gatan Digital Micrograph as software (both Gatan Inc.).
4
Nanomaterial Characterization by Electron Microscopy
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Average size, distribution, and morphology were investigated by electron microscopy (EM) with three modes: transmission (TEM), scanning (STEM), and high-resolution (HRTEM) using a JEOL 2100F (Tokyo, Japan) instrument operated at an accelerating voltage of 200 kV and equipped with a CMOS camera to acquire TEM and HRTEM images. Lattice fringes were measured from the fast Fourier transform of HRTEM images, using a Gatan Digital Micrograph (GATAN Inc., Pleasanton, CA, USA). STEM images were obtained using a high angular annular dark field (HAADF) field detector. The microscope is equipped with accessories for energy-dispersive X-ray spectroscopy (EDS) and electron energy loss spectroscopy (EELS) (EELS-GIF Tridiem GATAN). The elemental compositions were investigated by EDS and EELS to evaluate the atomic composition at the nanoscale using the mode STEM. EELS experiments were conducted in the STEM imaging mode using a spot size of 0.7 nm. The spectrometer aperture was 5 mm. The energy resolution measured by the FWHM of the zero-loss peaks was approximately 1.8 eV.
5
Nanoparticle Characterization via Microscopy and Spectroscopy
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The dimensions of the TiON/C-CNF1s and TiON/C-CNF2s were determined in the Fiji (ImageJ) [32 (link)] program (version 1.53o, Wayne Rasband and contributors, National Institutes of Health, USA) from calibrated micrographs. The nanoparticle size distribution (as equivalent radii) of TiON/C-CNF2s was determined by a custom-made program. XRD diffractogram was generated using CrystalDiffract®, a powder diffraction program for Mac and Windows (version 9.1.4(633), David Palmer, CrystalMaker Software Ltd., Oxford, England, www.crystalmaker.com , access date 23 June 2022) and EDXS spectrum was simulated with the DTSA-II program [33 (link)] (version Kelvin 2018-06-01, Nicholas W. M. Ritchie, Microanalysis Group, National Institute of Standards and Technology, Gaithersburg, MD, USA). At. % from STEM EDXS spectra were quantified using the Jeol AnalysisStation® program (version 3, 8, 0, 34, JEOL Ltd., Tokyo, Japan). At. % of elements obtained from EELS were calculated with Gatan Digital Micrograph program (version 2.32.888.0, Gatan inc., Pleasanton, CA, USA).
6
Characterizing Extracellular Vesicles by TEM
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For transmission electron microscopy, EVs from SCC-9 and LN1 cell lines were resuspended in PBS (2.5 × 10e8 particles per grid) and adsorbed on an Ultrathin Carbon Film/Haley Carbon 400 mesh copper grid positively charged with 15 mA for 25 s. The carbon grids were stained with 2% uranyl acetate and analyzed in the transmission electron microscope JEOL 1400 PLUS (JEOL Ltd) equipped with a tungsten filament and operated at an acceleration voltage of 120 kV. The images were acquired in an OneView camera (4K × 4K pixel) using the Software Gatan DigitalMicrograph (Gatan Inc).
7
Isolation and Visualization of Microvesicles
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Microvesicles were isolated by ultracentrifugation as described above and reconstituted in PBS. Samples were added onto a formvar-coated 200-mesh grid (Oxford Instruments) and incubated for 45 min in RT. The grid was dried and 1 % uranyl acetate was added for 10 s. Before analysis in a Tecnai G2 transmission electron microscope (FEI Company), the grid was dried for at least 15 min. ORIUS SC200 CCD was used as camera and Gatan Digital Micrograph as software (both Gatan Inc.).
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