Samples were prepared for electron microscopy analysis using a standard procedure [16] 16. Sabatini, DD • Bensch, K • Barrnett, RJ Cytochemistry and electron microscopy: The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation J Cell Biol. 1963; 17:19-58 Crossref Scopus (2029) PubMed Google Scholar and a transmission electron microscope (H-7650 and HT-7700, Hitachi High-Technologies, Tokyo, Japan) was used for observation. Scanning transmission electron microscopy (STEM)/energy-dispersive X-ray analysis was performed using an HT-7700 (Hitachi High-Technologies) to detect elements in high-density deposits.
Ht7700
Manufactured by Hitachi
Sourced in Japan, United States, Germany, China, United Kingdom
The HT7700 is a high-resolution transmission electron microscope (TEM) designed for materials analysis and characterization. It provides advanced imaging and analytical capabilities for a wide range of applications in materials science, nanotechnology, and life sciences. The core function of the HT7700 is to enable high-resolution, high-contrast imaging and elemental analysis of nanoscale structures and materials.
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Jem 2100f, by JEOL (10 mentions)
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Smartlab, by Rigaku (9 mentions)
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1 739 protocols using ht7700
1
Electron Microscopy Sample Preparation
2
Ultrastructural Analysis of dECM and Mitochondria
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For observing dECM, the dECM sample was fixed with 2.5% glutaraldehyde and dehydrated with an increase in ethanol concentration (50%, 75%, 80%, 95%, and 100%). The morphology of dECM samples was analyzed by scanning electron microscope (HT7700; Hitachi Hi-Tech, Tokyo, Japan).
For cultured cell samples, the conventional sample preparation process was adopted, including dehydration, immersion, embedding, ultra-thin section, and heavy metal staining. The morphology of mitochondria was observed by transmission electron microscope (Hitachi HT7700, Tokyo, Japan) at an accelerated voltage of 220kV.
For cultured cell samples, the conventional sample preparation process was adopted, including dehydration, immersion, embedding, ultra-thin section, and heavy metal staining. The morphology of mitochondria was observed by transmission electron microscope (Hitachi HT7700, Tokyo, Japan) at an accelerated voltage of 220kV.
3
Multitechnique Characterization of Materials
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Powder X-ray diffraction patterns (XRD) were measured using a Bruker AXS D8 Advance diffractometer (Billerica, MA, USA) in Bragg–Brentano geometry (Cu Kα radiation λ = 0.15406 nm). Transmission electron microscopy (TEM) (Hitachi HT7700, Ltd. Tokyo, Japan) images were taken with a Hitachi HT7700 transmission electron microscope (100 kV accelerating voltage). An Andor Shamrock 500i spectrometer (Andor Technology Ltd., Belfast, UK), coupled with a silicon iDus CCD camera, working as a detector, was used for the emission spectra measurements. The samples were excited by the use of the fiber-coupled, solid-state diode pumped (SSDP) 975 and 785 nm lasers, i.e., FC-975-2W (CNI; Changchun, China) and LW-785-120-C12-DM (Lambdawave, Wrocław, Poland), respectively.
In both cases, the beam spot sizes were ≈200 µm (Gauss profile), and the laser power was adjusted to ≈100 mW, for both excitation wavelengths, which corresponds to the power densities of ≈50 W cm−2. The luminescence decay curves were recorded using a 200 MHz Tektronix MDO3022 oscilloscope, coupled to the R928 PMT (Hamamatsu, Shimokanzo, Japan) and a QuantaMaster™ 40 spectrophotometer (Photon Technology International, Birmingham Rd, Birmingham UK). A tunable Opolette 355LD UVDM, nano-second pulsed laser, with a repetition rate of 20 Hz (Opotek Inc., Faraday Ave Suite E, Carlsbad, CA, USA), was used as the excitation source.
In both cases, the beam spot sizes were ≈200 µm (Gauss profile), and the laser power was adjusted to ≈100 mW, for both excitation wavelengths, which corresponds to the power densities of ≈50 W cm−2. The luminescence decay curves were recorded using a 200 MHz Tektronix MDO3022 oscilloscope, coupled to the R928 PMT (Hamamatsu, Shimokanzo, Japan) and a QuantaMaster™ 40 spectrophotometer (Photon Technology International, Birmingham Rd, Birmingham UK). A tunable Opolette 355LD UVDM, nano-second pulsed laser, with a repetition rate of 20 Hz (Opotek Inc., Faraday Ave Suite E, Carlsbad, CA, USA), was used as the excitation source.
4
Minimizing Electron Beam Damage in TEM Imaging
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TEM imaging was carried out via JEOL 1220 or Hitachi HT7700. Scanning transmission electron microscopy (STEM) imaging, spatially resolved EDS and EELS were conducted via Hitachi HD2300, respectively. For low loss EELS, 0.1 eV/channel dispersion was used. Microscopes were operated at a low energy of 80 keV to minimize electron beam damage to the cells. Graphene itself further protected the cells from beam damage by minimizing the radicals and conducting the electrons. During the course of the TEM imaging (0.1 seconds exposure for video recording), electron dose was kept lower than 100 e/nm2, which was discussed earlier by de Jonge and Ross.64 (link) The dose values are estimates based on experiments performed by Hitachi HT7700. For STEM imaging, the electron dose was kept lower than the critical dose reported by Kennedy et al.65 (link) During TEM imaging, both high and low magnification imaging were carried out on most of the cells on the grids, with the main aim of keeping the cells intact in the vacuum environment.
5
Transmission Electron Microscopy Characterization of Li2CO3-K2CO3 Nanofluids
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Material characterization was performed using transmission electron microscopy (Hitachi HT 7700). Samples from pure Li2CO3–K2CO3 and its nanofluids thermally cycled at 2 °C min−1 and 10 °C min−1 were dissolved in 200 proof ethyl alcohol for dilution while minimizing exposure to moisture. The samples were then poured onto to carbon-coated copper grid and the grids were then imaged using Hitachi HT 7700 Transmission electron microscopy. 100 kV voltage was applied between cathode and anode to avoid transmission of excessive energy and possible ionization of samples. The beam height and alignment were adjusted at 50 000× and all imaging was carried out below this magnification. This was done to avoid any diffraction resulting in shadow formation of particles that may look like nanostructure. 300 nm objective aperture was used to avoid stigmation. All images were taken in the high-resolution mode for better sizing and observation of structures.
6
Ultrastructural Analysis of C. vulgaris Exposed to Ag Ions and AgNPs-G
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The ultrastructural analysis of C. vulgaris treated with different concentrations of Ag ions and AgNPs-G for one day and one week was performed by TEM (Hitachi HT 7700 transmission electron microscopy) analysis.
Algae were centrifuged to remove culture media and then fixed with glutaraldehyde (2.5% in sodium cacodilate buffer 0.1 M, pH 7.2) for 2 h at 4 °C. Then, samples were washed twice for 15 min in sodium cacodilate buffer, postfixed in osmium tetraoxide (1% in sodium cacodilate buffer 0.1 M, pH 7.2) and washed twice for 30 min in deionised H2O. Samples were stained with 0.5% uranyl acetate o.n. (over night) at 4 °C. Samples were dehydrated in a graded series of ethanol, from 30% to 100%. After dehydration, samples were embedded in Spurr resin (TAAB, Berks, UK).
Ultrathin sections of 50 nm in thickness were then cut using an ultramicrotome PowerTome PT-PC (RMC, Tucson, AZ, USA). Sections were picked up in 200 mesh copper grids and examined under a Hitachi HT7700 transmission electron microscope (Tokyo, Japan) at 75 kV.
Samples were analyzed by energy-dispersive X-ray spectroscopy (EDX) microanalysis with the TEM module of the Auriga 405 microscope (Carl Zeiss AG, Oberkochen, Germany) for the elemental analysis of the electron-dense particles inside the cells.
Algae were centrifuged to remove culture media and then fixed with glutaraldehyde (2.5% in sodium cacodilate buffer 0.1 M, pH 7.2) for 2 h at 4 °C. Then, samples were washed twice for 15 min in sodium cacodilate buffer, postfixed in osmium tetraoxide (1% in sodium cacodilate buffer 0.1 M, pH 7.2) and washed twice for 30 min in deionised H2O. Samples were stained with 0.5% uranyl acetate o.n. (over night) at 4 °C. Samples were dehydrated in a graded series of ethanol, from 30% to 100%. After dehydration, samples were embedded in Spurr resin (TAAB, Berks, UK).
Ultrathin sections of 50 nm in thickness were then cut using an ultramicrotome PowerTome PT-PC (RMC, Tucson, AZ, USA). Sections were picked up in 200 mesh copper grids and examined under a Hitachi HT7700 transmission electron microscope (Tokyo, Japan) at 75 kV.
Samples were analyzed by energy-dispersive X-ray spectroscopy (EDX) microanalysis with the TEM module of the Auriga 405 microscope (Carl Zeiss AG, Oberkochen, Germany) for the elemental analysis of the electron-dense particles inside the cells.
7
Ultrastructural Analysis of Luteolin-Treated Pyogenes
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T. pyogenes cells cultured to the logarithmic phase were diluted to form the bacterial suspension (approximately 1×106 CFU/mL) and treated with luteolin (final concentration, 1/2 MIC). The bacterial suspensions were incubated on a shaker at 37 ºC and 150 rpm for 12, 24, and 36 h, respectively. The bacterial cells were harvested by centrifugation at 3000 rpm for 10 min at 4 ºC, washed thrice with PBS. Then the specimens were fixed to the fibrous carbon film and observed using TEM (HT-7700, Hitachi, Japan).
Furthermore, the ultrastructure of bacteria was analysed by ultramicrotomy observation. After the bacterial cells were harvested and washed, the specimens were subjected to fixation with 2.5% glutaraldehyde at 4 ºC overnight. The bacterial cells were then pre-embedded with agar, washed thrice with PBS (10 min per wash) and post-fixed with 1% osmium tetroxide for 1 h. Thereafter, the cells were dehydrated with dehydrant (50% ethanol, 75% ethanol, 80% acetone, 90% acetone, 95% acetone and twice at 100% acetone, each time for 15 min), and then embedded and soaked with epoxy 812. Finally, the specimens were sectioned with an ultramicrotome, stained with uranyl acetate, and observed using TEM (HT-7700, Hitachi, Japan).
Furthermore, the ultrastructure of bacteria was analysed by ultramicrotomy observation. After the bacterial cells were harvested and washed, the specimens were subjected to fixation with 2.5% glutaraldehyde at 4 ºC overnight. The bacterial cells were then pre-embedded with agar, washed thrice with PBS (10 min per wash) and post-fixed with 1% osmium tetroxide for 1 h. Thereafter, the cells were dehydrated with dehydrant (50% ethanol, 75% ethanol, 80% acetone, 90% acetone, 95% acetone and twice at 100% acetone, each time for 15 min), and then embedded and soaked with epoxy 812. Finally, the specimens were sectioned with an ultramicrotome, stained with uranyl acetate, and observed using TEM (HT-7700, Hitachi, Japan).
8
Characterization of Bi2MoO6/MXene Heterostructures
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The morphology of the prepared Bi2MoO6, MXene nanosheets, and Bi2MoO6/MXene heterostructures was observed through scanning electron microscope (SEM, Hitachi S4800), transmission electron microscope (TEM, Hitachi HT7700), and scanning transmission electron microscopy equipped with energy-dispersive X-ray spectroscopy (EDS) for elemental mapping (STEM, Hitachi HT7700). Zeta potentials were measured by a Marlvern laser particle size analyzer (ZS980). XRD patterns were performed using X’Pert-Pro MPD (PANalytical, the Netherlands) diffractometer with monochromatic Cu Ka radiation (λ = 1.5418 Å, with scan speed of 4° min−1). Raman spectra were conducted through Raman spectrometer (Renishaw 1000) with a 1 mW He–Ne laser (633 nm) as an irradiation source. XPS analysis was performed by ESCALAB 250 (ThermoFisher Scientific, USA). AFM image was collected using atomic force microscope (Dimension ICON).
9
Surface and Mechanical Characterization of Clad Layers
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Surface roughness of the samples was measured by a 3D profiler (Bruker, ContourTG-K0, Germany). Five measurements with a scan area of 0.09 × 0.12 mm2 were performed at random positions on 3 samples.
The thickness of the clad layer was thinned by Focused Ion Beam (FIB, FEI Helios 600i, America), with the parameter 30 kV, 0.79 nA (1000 nm), 0.23 nA (300 nm), 80pA (120 nm), and calibrated by Transmission Electron Microscope (TEM, Hitachi HT7700, Japan) Pt and C was selected as protection layer and calibrated by Transmission Electron Microscope (TEM, Hitachi HT7700, Japan). The surface composition was analyzed using X-ray Photoelectron Spectroscopy (XPS, Thermo Scientific Escalab 250X, America), with an Al-Kα X-ray source (1486.6 eV) at a take-off angle of 90°.
The Vickers hardness was measured on the polished samples through a microscopic Webster hardness Tester with a 500 N load and a dwell time of 15 s (diamond indenter φ 2.8 mm). An average of 12 points was tested for each sample; the deviation in these measurements was less than ±0.3 %.
The thickness of the clad layer was thinned by Focused Ion Beam (FIB, FEI Helios 600i, America), with the parameter 30 kV, 0.79 nA (1000 nm), 0.23 nA (300 nm), 80pA (120 nm), and calibrated by Transmission Electron Microscope (TEM, Hitachi HT7700, Japan) Pt and C was selected as protection layer and calibrated by Transmission Electron Microscope (TEM, Hitachi HT7700, Japan). The surface composition was analyzed using X-ray Photoelectron Spectroscopy (XPS, Thermo Scientific Escalab 250X, America), with an Al-Kα X-ray source (1486.6 eV) at a take-off angle of 90°.
The Vickers hardness was measured on the polished samples through a microscopic Webster hardness Tester with a 500 N load and a dwell time of 15 s (diamond indenter φ 2.8 mm). An average of 12 points was tested for each sample; the deviation in these measurements was less than ±0.3 %.
10
TEM Imaging of Microcapsules and Plantosomes
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High-magnification transmission electron micrographs of the microcapsules/plantosomes were acquired using a TEM from Hitachi, model HT7700 (Japan, Hitachi HT7700 02.05 software) at an accelerating voltage of 100 kV in high-contrast mode. The microcapsules/plantosomes (after chloroform evaporation) were first washed with water (suspension diluted 20 times with MilliQ-water, separation of microcapsules/plantosomes from water phase. Taking 200 µL of the separated microcapsules/plantosomes and diluting with 800 µL MilliQ) and deposited onto 200 mesh Formvar/carbon TEM grids (Ted Pella, 01800-F) and thoroughly air-dried. Then the lipid contents were removed by dipping the TEM grid into 2.5 mL of 100 mM NaOH solution for 3 min followed by thorough washing with MilliQ water (dipping into 2.5 mL MilliQ-water for 2 min and repeating a second time with fresh MilliQ-water) and air-dried before imaging. The images were taken without staining.
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