For SEM, cells were fixed with 2.5% glutaraldehyde solution for 2 h, rinsed with PBS for 5 min, and fixed in osmium tetroxide for 2 h. Samples were then dehydrated in a graded ethanol series over 30 min and dried in a critical point dryer. Samples were prepared by sputter coating with 1 nm to 2 nm gold-palladium and analysed using field-emission SEM (Hitachi, Tokyo, Japan). For TEM, cells were fixed in suspension at 37 °C in a 5% CO2 incubator. Cells (1 mL) were added to 9 mL of fixative (2.2% glutaraldehyde in 100 mM NaPO4 (pH 7.4)) and fixed for 2 h. Cells were post-fixed in 1% osmium tetroxide, stained en-bloc with 0.5% uranyl acetate in water, dehydrated in a graded ethanol series, embedded, and thin-sectioned. Sections were stained with 2% uranyl acetate in methanol for 20 min, followed by lead citrate for 5 min, and then observed using a Tecnai 12 electron microscope (FEI, Hillsboro, OR, USA) at 120 kV under low-dose conditions.
Field emission sem
Manufactured by Hitachi
Sourced in Japan
The Field-emission SEM is a scanning electron microscope that utilizes a field-emission electron source to generate a high-resolution, high-quality electron beam. This instrument is capable of producing detailed images of sample surfaces with nanometer-scale resolution.
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5 protocols using field emission sem
1
Ultrastructural Analysis of Cells
2
Scanning Electron Microscopy of LRCD-Encapsulated Compounds
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Hitachi field emission SEM was used to investigate the particle shape of the LRCD-encapsulated aromatic compounds (SEM S-4800, Hitachi, Tokyo, Japan). We examined the sample particles’ micromorphology and structure. The sample must be sprayed with a 1.0 kV accelerating voltage and magnified 1000 times [17 (link)].
3
Transmission and Scanning Electron Microscopy of Isolated TISs
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For transmission electron microscopy, isolated TISs were fixed in suspension at 37 °C in a 5% CO2 incubator and TISs containing the sample (1 mL) were added to 9 mL of fixative (2.2% glutaraldehyde in 100 mM NaPO4 [pH 7.4]) for 2 h. Samples were postfixed in 1% osmium tetroxide, stained en bloc with 0.5% uranyl acetate in water, dehydrated in a graded ethanol series, embedded, and thinly sectioned. The sections were stained with 2% uranyl acetate in methanol for 20 min, followed by lead citrate for 5 min, and then observed under a Tecnai G2 electron microscope (FEI, Hillsboro, OR, USA) at 120 kV under low-dose conditions. For SEM, cells were fixed with a 2.5% glutaraldehyde solution and osmium tetroxide for 2 h. Samples were then dehydrated in a graded ethanol series for 30 min, dried, prepared by sputter coating with 1–2-nm gold–palladium, and analyzed using a field-emission SEM (Hitachi, Tokyo, Japan).
4
Comprehensive Nanoparticle Characterization Protocol
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UV-vis spectroscopy (HP 8453, Agilent Technologies, Santa Clara, CA, USA) was used to characterize the absorption spectra of the sample in the wavelength ranges of 200–1,000 nm. The particle size was measured at ambient temperature using the intensity-based dynamic light scattering method. Field emission-SEM (Hitachi Ltd., Tokyo, Japan) was also employed to investigate the size and morphology of prepared samples. Transmission electron microscopy (TEM, AP Tech, McMinnville, OR, USA, Tecnai, G2 F30 S-Twin) was performed to confirm the existence of the core–shell nanostructure of prepared samples using an accelerating voltage of 300 kV. A Monora500i micro Raman spectrometer (ANDOR, Belfast, UK) was used to characterize the optical properties of these NPs. The Raman spectrograph employed a 1,200 g/mm grating and laser excitation at 632.8 nm with accumulation times of 5 seconds.
5
Comprehensive Material Characterization by Advanced Spectroscopy
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The morphology and microstructure of samples were examined by a Hitachi field-emission SEM. ATR-IR spectra were collected on the Spectrum 100 spectrometer with an ATR-IR signal collector. XPS analyses were conducted on a Thermo Fisher ESCALAB 250 photoelectron spectrometer at 1.2 × 10−9 mbar using an Al Kα X-ray beam (1486.6 eV). XPS spectra were charge corrected to the adventitious C 1 s peak at 284.6 eV. The C and O K-edge XANES spectra were collected at the BL12B beamlines of National Synchrotron Radiation Laboratory (Hefei, China). In operando Raman spectra were recorded using a LabRam HR Raman Spectrometer, with a laser wavelength of 532 nm.
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