The microstructure of the 3D printed scaffolds with differing numbers of layers were evaluated using scanning electron microscopy (SEM) (Hitachi SU8010). A total of 8 scaffolds were selected for SEM analysis: one per studied structure (i.e., lattice and staggered) and layer number (i.e., 4–10 layers) group; SEM images from cross-sections were taken. Each scaffold was coated with 10 nm of gold (Quorum Q150TES Sputter Coater) and then scanned under high vacuum at an accelerating voltage of 3.0 kV. Of note, for the 10-layer scaffolds, visualizing all the layers within a single image was not feasible due to limitations associated with the lowest SEM magnification. SEM images were used to measure microstructural parameters including the amount of penetration between layers (Δ0) as well as pore width (Px) and pore height (Pz) (or layer gap), which are displayed in the illustration in Fig 2 . Of note, penetration between layers pertains to the amount of diffusion between strands in subsequent layers. All strand diameters and distances between strands, as shown in Fig 2 , were also measured. ImageJ software (National Institutes of Health [30 (link)]) was used to measure the microstructural parameters. Measurements pertain to only one lay-down direction since SEM images were taken from one side only.
Q150t es sputter coater
Manufactured by Quorum Technologies
Sourced in United Kingdom, Germany
The Q150T ES sputter coater is a laboratory equipment used for depositing thin films on samples. It operates by sputtering material from a target onto the sample surface, creating a uniform coating. The core function of this device is to provide a controlled environment for thin film deposition, which is a fundamental process in various scientific and technological applications.
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Lab products found in correlation
Sigma hd vp fe sem, by Zeiss (5 mentions)
Merlin fesem electron microscope, by Zeiss (3 mentions)
Su8010, by Hitachi (2 mentions)
Supra field emission scanning electron microscope, by Zeiss (2 mentions)
Samdri 795 critical point dryer, by Tousimis (2 mentions)
Autoslice and view software, by Thermo Fisher Scientific (1 mentions)
Amira avizo software, by Thermo Fisher Scientific (1 mentions)
Auriga, by Zeiss (1 mentions)
Jem 1011 tem, by JEOL (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
S2400 microscope, by Hitachi (1 mentions)
Jsm 7001f, by JEOL (1 mentions)
Nova nanosem 450, by Thermo Fisher Scientific (1 mentions)
Tem image and analysissoftware ver 4, by Thermo Fisher Scientific (1 mentions)
Talos l120c, by Thermo Fisher Scientific (1 mentions)
Ceta 16m ccd camera, by Thermo Fisher Scientific (1 mentions)
Spurr s resin, by TAAB (1 mentions)
Fesem, by Thermo Fisher Scientific (1 mentions)
Su8010 sem, by Hitachi (1 mentions)
Dfc425, by Leica (1 mentions)
33 protocols using q150t es sputter coater
1
Microstructural Analysis of 3D Printed Scaffolds
2
Quantifying Osteoclast Resorption Activity
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Resorption pit areas were measured from Field Emission Scanning Electron Microscopy (FESEM) images with Merz grid analysis. After multinuclear cell counting, cells were detached from the slices by brushing, samples were dehydrated in ascending ethanol series and dried with a critical point drying equipment K850 (Quorum technologies, UK). Samples were coated with 5 nm platinum by Q150T ES sputter coater (Quorum Technologies) and viewed with Sigma HD VP FE-SEM (Carl Zeiss Microscopy GmbH, Germany). FESEM images were taken from three fields (voltage 5.0 kV, magnification 50×, area 0,035 cm2) from each bone slice, n = 3. The morphometric analysis of the pits was performed with ImageJ 1.49t software (NIH, USA) by superimposing a Merz grid with 80–88 points in semicircular lines over the image. Points in pits were counted and the proportion of resorption pits versus intact bone surface was counted. The proportion of resorbed area was normalized to multinuclear cell number (counting described above), and the average area resorbed by one cell was calculated.
3
Morphological Changes in Brevundimonas Biofilm
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The effect of RF-API and Chl-API on the morphology of Brevundimonas sp. ESA1 biofilm were investigated by scanning electron microscopy (SEM) (CamScan Apollo 300, Cambridge, UK). For the SEM analysis biofilms were grown in MtPs as it is described in Section 2.7. After the RF-API and Chl-API treatment (as described in Section 2.8) at 25 mW/cm2 for 95 min and 20 mW/cm2 for 60 min (irradiation conditions under which 3 log10 reduction was achieved), respectively, irradiated biofilms and dark controls were mechanically detached by scraping them of the MtP well walls by pipette tip, then 10 µl of each sample was transferred on a SEM specimen stub covered with copper foil tape, air-dried at room temperature and coated with 50 nm gold layer using Q150T ES sputter coater (Quorum Technologies, Lewes, England). The scanning was performed using an electron beam with an accelerating voltage of 20 kV.
4
Quantifying Bone Resorption via FESEM Imaging
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Resorption pit areas were measured from Field Emission Scanning Electron Microscopy (FESEM) images with Merz grid analysis. After multinuclear cell counting, cells were detached from the slices by brushing, samples were dehydrated in ascending ethanol series and dried with a critical point drying equipment K850 (Quorum technologies, UK). Samples were coated with 5 nm platinum by Q150T ES sputter coater (Quorum Technologies) and viewed with Sigma HD VP FE-SEM (Carl Zeiss Microscopy GmbH, Germany). FESEM images were taken from three fields (voltage 5.0 kV, magnification 50 ×, area 0.039 cm2) from each bone slice, n = 3. The morphometric analysis of the pits was performed with ImageJ 1.51n software (NIH, USA) by superimposing a Merz grid with 92–96 points in semicircular lines over the image. Points in pits were counted and the percentage of resorption pits versus intact bone surface was counted. The percentage of resorbed area was normalized to multinuclear cell number (counting described above), and the average area resorbed by one cell was calculated.
5
High-Resolution FIB-SEM Imaging of Resin-Embedded Tissues
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Resin-embedded tissue blocks were trimmed, mounted on SEM stubs, and then coated with a 5 nm platinum layer using a Q150T-ES sputter coater (Quorum Technologies, UK) before FIB-SEM volume imaging. Data was acquired using Scios Dual beam microscope (FIB-SEM) (Thermo Fischer Scientific). Electron beam imaging was acquired at 2 kV, 0.1 nA current, 1.9 × 1.9 nm pixel spacing, 7 mm working distance, 10 µs acquisition time, and 3072 × 2048 resolution using a T1 detector. SEM images were acquired every 20 nm. The working voltage of gallium ion beam was set at 30 kV, and 0.5 nA current was used for FIB milling. The specimens were imaged at 5 × 5 µm block face and 5 µm depth. FIB milling and SEM imaging were automated using the Auto Slice and View software (v4.1.1.1582, Thermo Fischer Scientific). SEM volume images were aligned and reconstructed using ImageJ (v. 1.8.0_172, NIH) with linear stack alignment, with SIFT and MultiStackRegistration plugins77 ,78 . Analysis and segmentation of SEM volume images were done using Amira-Avizo software (v2020.3.1, Thermo Fisher Scientific).
6
Structural Analysis of PVDF Nanocomposites
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A Field Emission Scanning Electron Microscope (FE-SEM, Auriga, Carl Zeiss, Oberkochen, Germany) operating with an accelerating voltage of 3 kV was used to assess the morphology of the PVDF nanocomposite films. A Quorum Technologies Q150T ES sputter coater (Laughton, East Sussex, UK) was used to metallize the PVDF films prior to SEM imaging with 20 nm of Cr, in order to prevent charging.
FT-IR measurements were performed using the same setup described in our previous work [22 ,23 (link)]. FT-IR measurements were carried out in the 4000–600 cm−1 range with a resolution of 1 cm−1.
X-ray powder diffraction (XRPD) measurements were performed using the same instrument and the same procedure described in [26 (link)]. Briefly, we used Cu Kα radiation (λ = 0.15418 nm) operating at 40 kV and 40 mA and operating in transmission mode. Data were collected in a 2θ angular range extending from 7° to 100° with a step size of 0.022° and 1 s counting time. Samples were prepared as capillaries loaded with nanostructures in powder form, obtained after three steps of dispersion using a high-shear mixer.
FT-IR measurements were performed using the same setup described in our previous work [22 ,23 (link)]. FT-IR measurements were carried out in the 4000–600 cm−1 range with a resolution of 1 cm−1.
X-ray powder diffraction (XRPD) measurements were performed using the same instrument and the same procedure described in [26 (link)]. Briefly, we used Cu Kα radiation (λ = 0.15418 nm) operating at 40 kV and 40 mA and operating in transmission mode. Data were collected in a 2θ angular range extending from 7° to 100° with a step size of 0.022° and 1 s counting time. Samples were prepared as capillaries loaded with nanostructures in powder form, obtained after three steps of dispersion using a high-shear mixer.
7
Critical Point Drying and SEM Imaging
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The ALD-HA samples were dehydrated in ascending ethanol series and dried with a critical point drying equipment K850 (Quorum Technologies, Lewes, UK). Samples were coated with 5 nm platinum by Q150T ES sputter coater (Quorum Technologies, Lewes, UK) and viewed with Sigma HD VP FE-SEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). FESEM images were taken with 5.0 kV voltage.
8
SEM Imaging of Listeria innocua
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For the preparation of scanning electron microscopy (SEM), L. innocua cells (1 × 109 CFU/mL) were incubated with nanoparticles and/or treated by AMFs and followed serial dilutions in sterile water. Then, 5 μL measurements of solutions were dropped onto the specimen stubs covered with copper foil tape and gently dried at room temperature. For the microscopy preparation, samples were sputter coated with a 25 nm gold layer using a Q150T ES sputter coater (Quorum Technologies, Laughton, UK). Twenty or more images per cell treatment were obtained using an Apollo 300 (CamScan, Cambridge, UK) scanning electron microscope operating at 15 kV.
9
Characterization of DPNs Embedded in Templates
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The size and shape of all silicon, PDMS, and PVA templates were characterized via Scanning Electron Microscope (Helios Nanolab 650). Ultra-high resolution SEM images were acquired at high vacuum conditions after 10 nm aurum coating using a Q150T ES sputter-coater (Quorum). DPNs still embedded within the PVA templates were observed using an A1 confocal microscope (Nikon) equipped with 63 × oil immersion objective. For EM characterization, a DPNs solution was dried on a carbon-copper grid and coated with 10–20 nm of carbon before Transmission Electron Microscope imaging (JEOL JEM 1011 TEM working at 100 KV). The ζ-potential was calculated using DLS (Malvern, UK).
10
Scaffold Pore Size Analysis via SEM
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Although printability was high, pore size of different scaffolds was verified using scanning electron microscopy (SEM) (Hitachi SU8010), which is a standard technique in the field. Each scaffold was coated with 10 nm of gold (Quorum Q150TES Sputter Coater) prior to the SEM analysis and then scanned under high vacuum at an accelerating voltage of 3.0 kV. The SEM images from various sites of cross sections were taken (Fig. 4 A, B). All scaffolds showed porous structures with well-defined geometry, quadrangular and interconnected pores, as well as good bond between layers. ImageJ [64 ] was used with SEM images to measure pore size, determined via the largest diameter circle, which fit between the strands. For this study, the range of pore sizes were 0.280–0.991 mm for lattice structures and 0.280–1.086 mm for staggered structures.
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