Q150t es

The spheres were air-dried on a glass slide and sputtered with platinum for 45 s in a sputter coater Q150T ES (Quorum Technologies, Ringmer, UK). Focused ion beam milling was performed using a Ga⁺ ion beam at 30 kV, 23 pA and 17 nm in diameter. The ion beam was used to cross-section individual particles within the sample. After FIB milling, the specimens were transferred to the scanning electron microscope and imaged as specified above.

CD9 positive fractions (Fractions 3–6) were thawed from a minus 80 °C freezer and prepared for SEM imaging. Both samples were diluted properly (at least 100 times) in PBS (pH 7.4) and volume of 10 μl of each dilution were deposited on a pure, thin glass substrate. The plates with fixed exosomes were stored in 4 °C temperature for gentle drying. So-prepared exosomes underwent immediate gold/palladium (80:20, 60 seconds) sputtering for scanning electron microscopy visualization. Structure of exosomes was analysed by scanning electron microscopy SEM (Jeol, JSM 7001 F TTLS). The etched surfaces were coated for 60 seconds with gold/palladium (80:20) using sputter coater/turbo evaporator (Quorum Technologies Q150T ES) in order to provide an electrically conductive thin film to reduce thermal damage and charging of the samples. The SEM micrographs were acquired by applying the accelerating voltage of 15 kV and SEI secondary electron methodology.

Heat-inactivated Delta variant of SARS-CoV-2 was diluted in 10 mM PBS (pH 7.6) to three concentrations (6.45 × 10³, 6.45 × 10⁴, and 6.45 × 10⁵ TCID₅₀/mL). The method for sample preparation was adopted from Haddad et al. (2020) (link) with slight modifications. The following steps were carried out in the fume hood. All samples were fixed by adding 2.5% glutaraldehyde (G5882, Sigma-Aldrich, USA) to the sample (1:1) for 1 h. 40 µL of the fixed sample was deposited on the pure carbon films (01840-F, Ted Pella Inc, USA) for 30 min, and a filter paper was used to carefully remove the excess liquid. The grids were then immediately stained with 1% osmium tetroxide (75632, Sigma-Aldrich, USA) and allowed to dry at room temperature. The three negatively stained carbon grids were then placed on the microscope stub using a double-sided adhesive tape and sputtered with a 10 nm-thick gold layer using a turbomolecular pumped coater Q150T ES (Quorum Technologies, UK) for 10 min, to reduce charging of the non-conductive samples. The micrographs were acquired using the scanning electron microscope crossbeam 540 (Carl Zeiss, Germany). The SEM allowed to observe samples under high vacuum pressure, and the images were acquired at magnifications ranging from 5 kX to 200 kX using an electron high tension (EHT) of 5 kV and a high-resolution energy selective backscattered (ESB) electron detector.

Micrographs were collected with a Mira3 system (Tescan) operated at 5 kV and a working distance of 6–7 mm. The samples were mounted on aluminium stubs using conductive carbon tape and coated with a 10 nm thick layer of platinum with a sputter coater (Quorum, Q150T ES). To image the exterior of the microparticles by SEM, the microparticles were dispersed in n-hexane and the suspension was drop cast onto a glass coverslip followed by drying with nitrogen. To image the interior of microparticles, they were cryo-fractured using the following protocol: (i) the microparticles in n-hexane were drop cast on a coverslip, (ii) embedded in cellulose butyrate acetate resin (10 wt.% cellulose butyrate acetate dissolved in ethyl acetate), (iii) transferred into a dry nitrogen atmosphere, (iv) frozen in liquid nitrogen for 5 min, (v) mechanically crushed.

The hydrogels were washed 2 × 5 min in 0.1 M Sorensen´s buffer (pH 7.4) (S/3760/60 Fisher Scientific and S/4520/60, Fisher Scientific) and fixed in approximately 10 times the sample volume of “SEM fix” (0.1 M Sorensen´s phosphate buffer pH 7.4, 2% formaldehyde (P001, TAAB) and 2% glutaraldehyde (18427, Ted Pella Inc)) at room temperature for 20 min, followed by washing 2 × 5 min in 0.1 M Sorensen´s buffer pH 7.4. Samples were then dehydrated in a graded series of ethanol (50%, 70%, 80%, 90%, and twice in 100%) and subsequently critical point dried before being mounted and sputter coated with 5–10 nm Pt/Pd (80:20) (MN70-PP5708, Micro to Nano) on a turbomolecular pump coater (Q150T ES, Quorum). As cryogels were already freeze dried, they were directly mounted and coated with 5–10 nm Pt/Pd (80:20) and all samples were examined in a JSM-7800F FEG-SEM (Jeol).

The morphologies of the dried cellulose hydrogels were characterized by field emission scanning electron microscopy (FE-SEM) (TESCAN MAIA 3; Oxford instrument, Abingdon, UK) at an accelerating voltage of 15 kV with a 5 nm iridium coating (Q150 T ES; Quorum Technologies, Lewes, UK).

SEM analyses were carried out by using a Tescan Vega 3 LM scanning electron microscope equipped with an Apollo X detector and a Microanalysis TEAM Energy Dispersive Spectrometry (SEM/EDS: TESCAN, Brno, Czech Republic). The samples were metalized with a 25–50 nm layer of Au by cathodic sputtering (Quorum Q150T ES). Microphotographs were acquired with an optimized ratio between back scattered and secondary electrons in order to preserve chemical and morphologic information.
The starting materials for the bioassays were SHS slags that had been hand-ground and sieved to separate the fractions, with grain sizes of <10 μm, <3 μm and <2 μm, referred to as samples A, B, and C, respectively. A specimen of pure chrysotile, previously characterised¹³ , was named sample D. The bulk composition of 5 slags was analysed by LiB Fusion followed by Inductively Coupled Plasma Mass Spectrometry (ICP MS) analysis.