Pristine PSNPLs (PP-008-10) and yellow fluorophore-conjugated PSNPLs (y-PSNPLs) (FP-00552-2) were purchased from Spherotech (Chicago, IL, USA). The pristine form of polystyrene (PS) particles was used for all the experiments carried out except for those in which the fluorescent marker was required, such as the visualization and the quantification of PS particles internalization by confocal microscopy and flow cytometry, respectively. PSNPLs used for the assays were characterized using Zetasizer and transmission electron microscopy (TEM) methodologies. To that purpose, the obtained dispersions were diluted to a final concentration of 100 μg/mL in distilled water. The hydrodynamic size and the Z-potential values for PSNPLs and y-PSNPLs dispersions were determined in a Malvern Zetasizer Nano ZS zen3600 device (Malvern, UK). All the parameters for each sample were measured in triplicates. TEM grids were dipped into PSNPLs and y-PSNPLs dispersions and visualized on a JEOL JEM-1400 instrument (JEOL LTD, Tokyo, Japan). To determine the mean size, 100 randomly selected PSNPLs were measured using an Image J software with the Fiji extension.
Jem 1400 instrument
Manufactured by JEOL
Sourced in Japan
The JEM-1400 is a transmission electron microscope (TEM) manufactured by JEOL. It is designed to provide high-quality imaging and analysis of a wide range of samples at the nanoscale level. The JEM-1400 uses an electron beam to interact with the specimen, allowing for the visualization and characterization of micro- and nanostructures.
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Lab products found in correlation
Pristine psnpls pp 008 10, by Spherotech (2 mentions)
Yellow fluorophore conjugated psnpls y psnpls fp 00552 2, by Spherotech (2 mentions)
Zetasizer nano zs zen3600 device, by Malvern Panalytical (2 mentions)
Tem 2011 200 kv instrument, by JEOL (1 mentions)
Inca detector, by Oxford Instruments (1 mentions)
D8 discover diffractometer, by Bruker (1 mentions)
D8 advance x ray diffractometer, by Bruker (1 mentions)
Tensor 27 ft ir spectrophotometer, by Bruker (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
Ultracut e ultramicrotome, by Leica (1 mentions)
Integra prima microscope, by NT-MDT (1 mentions)
Nova spm software, by NT-MDT (1 mentions)
Carbon formvar coated copper grids, by Ted Pella (1 mentions)
Nsg01, by NT-MDT (1 mentions)
Jem 2100f microscope, by Ametek (1 mentions)
11 protocols using jem 1400 instrument
1
Characterization of Polystyrene Nanoparticles
2
Silver Nanoparticle Interactions with Polystyrene
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To analyze the potential interactions between silver and PSNPs, after AgNPs/PSNPs, or AgNO3/PSNPs treatments the samples were visualized by TEM. For this purpose, AgNPs or AgNO3 were incubated with PSNPs in a distilled water dilution at a final concentration of 5 µg/mL AgNPs or AgNO3, and 10 or 100 µg/mL PSNPs for 3 h at room temperature. Then, carbon-coated TEM grids were dipped into the samples and visualized on a JEOL JEM-1400 instrument (JEOL LTD, Tokyo, Japan). To demonstrate that silver materials were on the polystyrene surface, both AgNPs/PSNPs and AgNO3/PSNPs samples were analyzed by transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy (TEM-EDX) with a TEM JEOL-2011 (200 kV) instrument (JEOL LTD, Tokyo, Japan) combined with an INCA detector (Oxford Instruments, United Kingdom).
3
Characterization of Pristine and Fluorescent Polystyrene Nanoparticles
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Pristine PSNPLs (PP-008-10) and yellow fluorophore-conjugated PSNPLs (y-PSNPLs) (FP-00552-2) were purchased from Spherotech (Chicago, USA). Pristine PSNPLs were used in all experimental work except for the quantification of PS nanoparticles internalization by flow cytometry, for which the fluorescently labeled particle was required. Nanoplastic characterization was performed using a Zetasizer device and transmission electron microscopy (TEM). For that purpose, the PSNPLs dispersions were diluted to a final concentration of 100 μg/mL in distilled water or in a culture medium (DMEM). The hydrodynamic size and Z-potential parameters were analyzed in triplicates with a Malvern Zetasizer Nano ZS zen3600 device (Malvern, UK), applying dynamic light scattering (DLS) and electrophoretic light scattering (ELS) approaches. On the other hand, carbon-coated TEM grids were dipped into the PSNPLs dispersions and visualized on a JEOL JEM-1400 instrument (JEOL LTD, Tokyo, Japan). To determine the mean particle size, 100 randomly selected PSNPLs were measured using Image J software with the Fiji extension.
4
Structural and Textural Analysis of Materials
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The materials structure was evaluated by X-ray diffraction using a Bruker D8 Discover diffractometer (Bruker, Billerica, MA, USA) with Cu Kα radiation (λ = 1.54 Å). The XRD patterns were recorded in a 2θ scan range from 15° to 70° with a scan speed of 0.5 or 1 °/min. Phase identification and crystallite size fitted were carried out using Diffrac.suite Eva software, supported by the Power Diffraction File Database.
Textural properties were evaluated by nitrogen adsorption and desorption, and isotherm experiments were accomplished in a porosimeter Micromeritics ASAP 2000 instrument (Micromeritics, Norcross, GA, USA). Samples were previously degassed at 130 °C for 24 h under vacuum (p < 10−2 Pa). The specific surface area of the synthetized materials was calculated using the Brunauer, Emmett and Teller (BET) equation in the linear region (0.05 < Po < 0.22).
The TEM images were recorded in a JEOL JEM 1400 instrument (JEOL USA, Inc, Peabody, MA, USA) and assembled with a charge-coupled camera device (Zeiss, Oberkochen, Baden-Württemberg, Germany). Samples were previously suspended in ethanol and deposited on a copper grid.
SEM-EDX micrographs were acquired in a JEOL-SEM JSM-7800 LV scanning microscope.
Textural properties were evaluated by nitrogen adsorption and desorption, and isotherm experiments were accomplished in a porosimeter Micromeritics ASAP 2000 instrument (Micromeritics, Norcross, GA, USA). Samples were previously degassed at 130 °C for 24 h under vacuum (p < 10−2 Pa). The specific surface area of the synthetized materials was calculated using the Brunauer, Emmett and Teller (BET) equation in the linear region (0.05 < Po < 0.22).
The TEM images were recorded in a JEOL JEM 1400 instrument (JEOL USA, Inc, Peabody, MA, USA) and assembled with a charge-coupled camera device (Zeiss, Oberkochen, Baden-Württemberg, Germany). Samples were previously suspended in ethanol and deposited on a copper grid.
SEM-EDX micrographs were acquired in a JEOL-SEM JSM-7800 LV scanning microscope.
5
Characterization of Nanocomposite Materials
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The size distribution and stability of the nanocomposites were determined using dynamic light scattering (DLS) and zeta potential measurements, which were measured on a nanoPartica Horiba SZ-100 (Japan). Fourier-transform infrared (FTIR) spectra were obtained using a Bruker Tensor 27 FTIR spectrophotometer (Germany). X-ray diffraction (XRD) patterns were collected using a Bruker D8 Advance X-ray diffractometer. The morphology of the nanocomposites was investigated using transmission electron microscopy (TEM) with a JEOL JEM-1400 instrument. Thermal analysis, including thermogravimetry analysis (TGA) and differential scanning calorimetry (DSC), was conducted using a LabSys Evo 1600 thermal analyzer (SETARAM, France) under atmospheric conditions from 25 to 800 °C with a heating rate of 10 °C/min.
6
Characterizing Protein Nanoparticle Microstructures
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The microstructures of PPI and PPIH were determined by transmission electron microscopy (TEM) according to Wang et al. [43 (link)]. The TEM images of PPI and PPIH were determined using a JEM-1400 instrument (JEOL Co. Ltd., Tokyo, Japan) operated at 100 keV. A 300-mesh formvar carbon-coated copper grid was used as the support substrate. The sample was prepared by dropping 10 μL of PPI and PPIH solutions onto the grids and blotting the excess sample after 3 min. Positive staining was obtained by adding uranyl acetate to the sample solutions on the TEM grids. Excess uranyl acetate was blotted and then allowed to air-dry. By using Lispixl (NIST freeware), the statistics describing the size distribution of the observed colloids were analyzed. Each sample was analyzed in triplicate.
7
Quantitative Image Analysis Techniques
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Methods for these techniques have been published (Ameen et al., 2001 (link); Mashukova et al., 2012 (link)). Immunofluorescence using phosphoepitope antibodies was performed after fixation in trichloroacetic acid (Hayashi et al., 1999 (link)). All immunofluorescence images were collected with a Leica SP5 confocal microscope, and semiquantitative image analysis was performed with Leica software as described later. Electron microscope samples were prepared by standard techniques, and images were obtained with a Jeol JEM-1400 instrument, using Gatan software.
For fluorescence image analysis, pixel quantification was performed as described previously (Wald et al., 2011 (link)). Briefly, images were collected, avoiding pixel saturation in the channel to be measured. ROIs matching the size of positive signal images were defined and used throughout the sampling. Examples of ROIs are shown inFigures 5A , 6A , and 7C (yellow squares). The analyses were conducted blindly, and ROIs were positioned on the desired region of cells (e.g., apical edge, cytoplasm, or nucleus) in the 4′,6-diamidino-2-phenylindole (DAPI) channel to randomize the sampling. One average of pixel values in the ROI per cell was collected and used for statistics, which comprised several cells from different animals.
For fluorescence image analysis, pixel quantification was performed as described previously (Wald et al., 2011 (link)). Briefly, images were collected, avoiding pixel saturation in the channel to be measured. ROIs matching the size of positive signal images were defined and used throughout the sampling. Examples of ROIs are shown in
8
TEM Imaging of Fixed Cells
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For our TEM study, cells were harvested by centrifugation (1000×g, 2 min), and fixed with 1.7% glutaraldehyde in 50 mM sodium cacodylate buffer (pH 7.0) for 2 h and then post-fixed in 2% osmium tetroxide in the same buffer for 2 h at room temperature. After dehydration in an ethanol series, fixed cells were embedded in Spurr’s resin. Ultrathin sections (80 nm thick) were cut with a diamond knife on an ULTRACUT E ultra-microtome (Leica, Wetzlar, Germany) and mounted on Formvar-coated grids. Sections were stained with 4% uranyl acetate for 18 min and 0.4% lead citrate solution for 7 min at room temperature and observed on a JEM-1400 instrument (JEOL, Tokyo, Japan) at 120 kV.
9
Multimodal Characterization of Purified Proteins
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Analysis of particle size by dynamic light scattering method was performed using a Zetasizer NanoS90 particle size analyzer (Malvern).
Atomic force microscopy was performed using an Integra Prima microscope and Nova SPM software (NT-MDT, Moscow, Russia). The scanning was performed in semi contact mode using gold cantilever NSG01 (NT-MDT).
Electron microscopy was performed on a JEM 1400 instrument (JEOL, Tokyo, Japan). Purified proteins were placed on carbon-formvar-coated copper grids (TED PELLA, Redding, CA, USA) and stained with 1% (w/v) uranyl acetate in methanol. The average size of the particles was determined using 10 particles.
Atomic force microscopy was performed using an Integra Prima microscope and Nova SPM software (NT-MDT, Moscow, Russia). The scanning was performed in semi contact mode using gold cantilever NSG01 (NT-MDT).
Electron microscopy was performed on a JEM 1400 instrument (JEOL, Tokyo, Japan). Purified proteins were placed on carbon-formvar-coated copper grids (TED PELLA, Redding, CA, USA) and stained with 1% (w/v) uranyl acetate in methanol. The average size of the particles was determined using 10 particles.
10
Nanoparticle Size Characterization by TEM
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Size distribution was confirmed by using TEM. The samples were put on carbon Formvar-coated grids, negatively stained with uranyl acetate 1.5%, and observed using a JEOL JEM-1400 instrument (JEOL, Tokyo, Japan) (120 kV).
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