The Scanning Electron Microcopy (SEM) method was used to observe bacterial cell morphology. Each bacterial culture suspension was adjusted to final concentration of 3 × 106 CFU/mL in Mueller-Hinton broth with the O. indicum young fruit extract (OIYF) at a concentration of 5 mg/mL and without extract (control culture) in parallel. The cell suspensions were incubated at 37 °C for 15 h and were harvested for Scanning Electron Microscopy sample preparations. The bacterial cells were washed three times with sterile deionized water at 2000× g centrifugation for 10 min. The cell pellet was collected and fixed by 10% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and 2.5% gluten-aldehyde (Sigma-Aldrich, St. Louis, MO, USA) solution for 30–60 min. The samples were further dehydrated by a serial concentration gradient of 30%, 50%, 70%, 80%, 90%, and 100% ethanol solutions and acetone. The dehydrated cells were transferred to clean glass coverslip, dried and coated by Sputter coater Q150R ES (Quorum, Tokyo, Japan). All samples were kept dried in CO2 critical point dryer until they were examined by ultra-high Resolution Scanning Electron Microscope Hitashi SEM SU8020 (Hitachi High-Tech, Tokyo, Japan). Bacterial cell morphological changes were observed.
Q150r es sputter coater
Manufactured by Quorum Technologies
Sourced in United Kingdom
The Q150R-ES is a sputter coater designed for depositing thin metallic films onto samples. It features a rotary-pumped vacuum system, with a base pressure of 5 x 10-6 mbar, and a sputtering chamber that can accommodate samples up to 150 mm in diameter. The Q150R-ES is capable of sputtering a variety of target materials, including gold, silver, and platinum, at user-selectable deposition rates.
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Lab products found in correlation
Vega3 microscope, by TESCAN (10 mentions)
Ultracentrifuge, by Beckman Coulter (3 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Carbon adhesive pads, by Agar Scientific (2 mentions)
Flexsem 1000, by Hitachi (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Rm2255, by Leica (1 mentions)
Aluminium stub, by Agar Scientific (1 mentions)
Trypsin, by Bioidea (1 mentions)
Karnovsky fixative, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
Zen 2 blue edition, by Zeiss (1 mentions)
Lsm 710 axioobserver, by Zeiss (1 mentions)
Ec plan neofluar 10 0.30m27 objective, by Zeiss (1 mentions)
Evo hd15, by Zeiss (1 mentions)
Glutaraldehyde solution, by Carl Roth (1 mentions)
Flexsem 1000 scanning electron microscope, by Hitachi (1 mentions)
Whatman filter paper, by Cytiva (1 mentions)
Quanta 450 feg, by Thermo Fisher Scientific (1 mentions)
Swinnex filter holder, by Merck Group (1 mentions)
24 protocols using q150r es sputter coater
1
Scanning Electron Microscopy of Bacterial Morphology
2
Tablet Microstructure Evaluation via SEM-BSE
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The inner microstructure of tablets was evaluated in the form of a quadratic mean of area equivalent diameter (d Avg ) of CaHPO 4 •2H 2 O particles/aggregates on the tablet radial cross-section areas. Tablets were, for this purpose, incorporated into paraffin wax matrices and cut on rotary microtome RM2255 (Leica Mikro spol. s.r.o., CZ), so the resulting cross-sections are flat and smooth [32] (link). Samples prepared in this way were subsequently sputter coated with 2 nm of platinum (sputter coater Q150R ES, Quorum Technologies Ltd, UK) and analysed using the SEM with detection of backscattered electrons (BSE). Here, the use of CaHPO 4 •2H 2 O as an excipient in model binary formulations and also as the primary source of the microstructural data can be justified. Thanks to its chemical nature, CaHPO 4 •2H 2 O provides a different contrast than ibuprofen during the BSE detection which is necessary for further image analysis. While the SEM-BSE was the most suitable method in the presented model system, other imaging methods such as scanning Raman spectroscopy or SEM-EDS could also be used for different systems as the input data for the subsequent image analysis, depending on the specifics and limitations of the tested systems.
3
Decellularized GOM Ultrastructure Analysis
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To evaluate the ultra-architecture of the decellularized
GOM, we performed scanning electron microscopy
(SEM). One part of each decellularized GOM was
fixed with 2.5% glutaraldehyde (Sigma-Aldrich,
St. Louis, MO, USA) in 0.2M PBS at pH=7.4 for 2
hours at 4°C. Subsequently, they were dehydrated in
an increasing graded series of ethanol (50-100%).
Finally, the samples were dried at the critical point
and coated with gold by Q150R- ES sputter coater
(QuorumTechnologies, UK); then, they were observed,
and photography was taken by a VEGA3 microscope
(TESCAN, Czech Republic).
GOM, we performed scanning electron microscopy
(SEM). One part of each decellularized GOM was
fixed with 2.5% glutaraldehyde (Sigma-Aldrich,
St. Louis, MO, USA) in 0.2M PBS at pH=7.4 for 2
hours at 4°C. Subsequently, they were dehydrated in
an increasing graded series of ethanol (50-100%).
Finally, the samples were dried at the critical point
and coated with gold by Q150R- ES sputter coater
(QuorumTechnologies, UK); then, they were observed,
and photography was taken by a VEGA3 microscope
(TESCAN, Czech Republic).
4
SEM Sample Preparation Protocol
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SEM analysis was performed using a 7800F SEM instrument (JOEL Ltd., Tokyo, Japan). Therefore, samples were positioned on aluminium stubs (Agar Scientific Ltd., London, UK) using carbon adhesive pads (Agar Scientific Ltd., UK). Afterwards, they were coated with a thin layer of gold (~10 nm) using a Q150RES sputter coater (Quorum Technologies Ltd., Laughton, UK).
5
Scaffold Preparation for SEM Imaging
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Frozen decellularized scaffolds were lyophilized overnight (Christ Alpha 2–4LD-plus, Osterode am Harz, Germany) to prepare for SEM. The samples were coated with a thin layer of gold using Q150R-ES sputter coater (Quorum Technologies, UK) and imaged by a VEGA3 microscope (TESCAN, Czech Republic).
6
Characterization of TQ-Free Scaffolds
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SEM images were prepared by lyophilizing TQ-free scaffolds. Gold replica was prepared using Q150R-ES sputter coater (Quorum Technologies, London, UK) and imaged using an VEGA3 microscope (TESCAN, Brno, Czech Republic) at 10 kV accelerating voltage. The SEM images were analyzed by ImageJ software to estimate the pore size. The porosity was measured by the liquid exchange method.
The degradation rate was measured by incubating the scaffolds in 0.01% trypsin (Bioidea, USA) and weighting them on days 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, and 56. The hydration rate was evaluated by immersing lyophilized TQ-free and loaded scaffolds in distilled water and weighting them at the beginning as well as at intervals of 5, 15, 30, 60, 120, and 240 s. To evaluate the impact of TQ loading on mechanical strength, the scaffolds at a size of 10 × 10 × 3 mm3 and the same porosity were exposed to a mechanical resistance determination device (Zwick/Roell, Germany) at a speed of 0.5 mm/min.
The degradation rate was measured by incubating the scaffolds in 0.01% trypsin (Bioidea, USA) and weighting them on days 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, and 56. The hydration rate was evaluated by immersing lyophilized TQ-free and loaded scaffolds in distilled water and weighting them at the beginning as well as at intervals of 5, 15, 30, 60, 120, and 240 s. To evaluate the impact of TQ loading on mechanical strength, the scaffolds at a size of 10 × 10 × 3 mm3 and the same porosity were exposed to a mechanical resistance determination device (Zwick/Roell, Germany) at a speed of 0.5 mm/min.
7
Scaffold Microarchitecture Evaluation by SEM
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In order to evaluate the microarchitecture of the decellularized scaffolds, scanning electron microscopy (SEM) was performed. The samples were fixed in 2.5% glutaraldehyde for 24 h at 4 °C, dehydrated in increasingly graded ethanol, and dried with increasingly graded hexamethyldisilazane. Finally, the samples were covered with gold using Q150R- ES sputter coater (Quorum Technologies, UK) and imaged by a VEGA3 microscope (TESCAN, Czech Republic).
8
Ultrastructure Analysis of Decellularized GOM
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To evaluate the ultra‐architecture of the decellularized GOM, scanning electron microscopy was applied. The sample pieces were fixed with Karnovsky fixative (Sigma‐Aldrich, St. Louis MO, USA) then dehydrated in an increasing graded series of ethanol (50%–100%). They were dried by incubating the samples in a gradually increasing concentration of HMDS. Finally, the pieces were coated with gold by Q150R‐ES sputter coater (Quorum Technologies, UK); they were then observed and imaged by a VEGA3 microscope (TESCAN, Czech Republic).
9
Ultrastructural Assessment of Decellularized Tissues
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For ultrastructural assessment, frozen decellularized samples were lyophilized overnight (Christ Alpha 2–4 LD-plus, Osterode am Harz, Germany). Sample preparation for intact ovaries and cell-containing scaffolds was based on a previously described method used by Kashi et al. [40 ]. Briefly, the samples were fixed using 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) plus 4% formaldehyde (Sigma-Aldrich, USA) in 0.1 M PBS (pH 7.4) at 4 °C overnight and then gradually dehydrated via an increasing graded series of ethanol. After that, the samples were immersed respectively into 1:2 and 2:1 Hexamethyldisilizane (HMDS; Merck, Kenilworth, NJ, USA): absolute ethanol for 20 min and then 100% HMDS solution overnight to air-dry in a fume hood. The samples were coated with a thin layer of gold, using Q150R- ES sputter coater (Quorum Technologies, London, UK) and imaged using an VEGA3 microscope (TESCAN, Brno, Czech Republic) at 10 kV accelerating voltage.
10
Pollen Morphology Characterization by SEM
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Size and shape of four selected pollen species, Iva xanthiifolia, Populus nigra italic, Populus tremuloides, and Populus deltoids (all from: Sigma-Aldrich, St. Louis, MO, USA) were examined through an electron microscope, the EVO HD 15 (Zeiss, Jena, Germany). Samples were sputtered with a thin gold layer using a Q150R ES sputter coater (Quorum technologies, Houston, TX, USA) before imaging at 5 kV and 7.5 mm working distance. A LSM 710 Axio Observer (Zeiss, Jena, Germany) visualized autofluorescence at a wavelength of 488 nm using an EC-Plan-Neofluar 10×/0.30M27 objective (Zeiss, Jena, Germany). ZEN 2 blue edition (Zeiss, Jena, Germany) was used as computer software. Additional wavelengths (405 & 561 nm) were applied when examining the ointment samples. For SEM imaging, MucilAir™ cell inserts were dehydrated with an ethanol (VWR Radnor, PA, USA) series at room temperature (RT) (70-80-90-96 (two times) and 100% for 10 min each), followed by sputter coating.
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