For the light microscopy imaging of the microcapsules, YS2-H optical, Nikon (Tokyo, Japan) was used, while for the scanning electron microscopy, and confocal imaging as well as the surface composition measurements Zeiss Neon 40EsB FIBSEM (Tescan, Brno, Czech Republic), UltraVIEW Vox, Perkin Elmer (Waltham, MA, USA), and Oxford Instruments, Aztec X-Act (Abingdon, U.K) were used as per our well-established procedures [32 (link),33 (link),34 (link)]. In brief, for the YS2-H optical imaging, dry microcapsules were placed on a glass slide and multiple images taken at different angles. The best image with best resolution was selected and presented. For the Zeiss Neon 40EsB FIBSEM scanning electron microscopic imaging, microcapsules were dried then coated with platinum, and using laser-guided pen, multiple images were taken. The best images with clear morphology relevant to specific desired magnifications were presented. For the UltraVIEW Vox confocal imaging complemented and equipped with a Yokogawa CSU-X1 confocal scanning unit, microcapsules with stained cells were imaged and multiple images taken, with the best resolution being presented.
Neon 40esb fib sem
Manufactured by Zeiss
Sourced in Germany
The Neon 40EsB FIB-SEM is a focused ion beam scanning electron microscope (FIB-SEM) system designed for high-resolution imaging and precise sample preparation. It combines a scanning electron microscope (SEM) with a focused ion beam (FIB) column, enabling both imaging and site-specific milling or deposition capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Incax act, by Oxford Instruments (3 mentions)
Molecular grade ethanol, by Merck Group (1 mentions)
Ultraview vox, by Yokogawa (1 mentions)
Ultraview vox, by PerkinElmer (1 mentions)
Csu x1 confocal scanning unit, by Yokogawa (1 mentions)
Zetasizer 3000hsa, by Malvern Panalytical (1 mentions)
Dsc 6000, by PerkinElmer (1 mentions)
Rectapur vwr, by Avantor (1 mentions)
Zetasizer nano s, by Malvern Panalytical (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
12 protocols using neon 40esb fib sem
1
Multimodal Microscopic Imaging of Microcapsules
2
Ethanol-Based SEM Preparation of AR42J-B13 Cells
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After five days of growth on etched coverslips in duplicate wells, AR42J-B13 cells were rinsed with PBS and immersed in an incremental gradient of 30, 50, 70, 90 and 100% molecular grade ethanol (Sigma), diluted in PBS. Samples were then coated with platinum and visualised with the assistance of Elaine Miller at Curtin University’s Microscopy and Microanalysis Facility on a Zeiss Neon 40EsB FIBSEM (Zeiss Australia, North Ryde, NSW, Australia).
3
Freeze Drying Surface Morphology Imaging
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Samples were freeze dried using Dynavac FD3 Freeze Dryer (Dynapumps, Seven Hills, Australia) for 48 h on −45 °C under vacuum. Afterwards, SEM (Zeiss Neon 40EsB FIBSEM, Carl Zeiss Microscopy GmbH, Jena, Germany) was utilised for surface morphology imaging [58 (link)].
4
Microcapsule Surface Morphology and Chemistry
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The surface morphology of the microcapsules was examined using SEM (Zeiss Neon 40EsB FIBSEM; Carl Zeiss AG) with 0.8 nm calibrated resolution. The chemical characterization of the microcapsules was examined using EDXR (AztecEnergy EDS Analysis Software, Oxford Instruments, Oxfordshire, UK). Electron micrographs of G and G-DCA-SA microcapsules were obtained using SEM, and their chemical characterization was obtained using EDXR. The samples were mounted on a glass stub with double-sided adhesive tape and coated under vacuum with platinum (5 nm) in an argon atmosphere prior to examination. Micrographs with different magnifications were recorded to study the morphological and surface characteristics of the microcapsules. Multiple images at various scales and angles were taken, and those that best captured the details of the surface morphological changes were used.
5
Morphological and Chemical Analysis of Microcapsules
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The surface morphology of the microcapsules was examined using SEM (Neon 40EsB FIB-SEM; Zeiss, Oberkochen, Germany) with 0.8 nm calibrated resolution. The chemical characterization of the microcapsules was examined using EDXR (INCA X-Act; Oxford Instruments, Abingdon, United Kingdom). Electron micrographs of PB-loaded microcapsules and empty SA microcapsules were obtained using SEM, and their chemical characterization was obtained using EDXR. The samples were mounted on a glass stub with double-sided adhesive tape and coated under vacuum with platinum (5 nm) in an argon atmosphere before examination. Micrographs with different magnifications were recorded to study the morphological and surface characteristics of the microcapsules.
6
Characterizing Microcapsules via Microscopy
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Morphological characterization was accessed by using Nikon H550S optical microscopy (OM) and scanning electron microscope (SEM) (Neon 40EsB FIB-SEM; Zeiss, Oberkochen, Germany). For SEM, the microcapsules were freshly made, dried and mounted on a glass slide stub with double-sided adhesive tape and coated under vacuum in an argon atmosphere with 5 nm platinum before examination. Elemental distribution present on the microcapsules’ surface was analysed by energy dispersive X-ray spectrometry (EDXR) (INCA X-Act; Oxford Instruments, Abingdon, UK) [28 (link)].
7
Microcapsule Characterization Protocol
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All microcapsules (both formulations) were prepared and analysed within 48 hours. The morphological analysis and size of microcapsules were undertaken by using OM, SEM and EDS. The microcapsules’ diameters were calculated with the help of the software ToupTek which is provided within the instrument.
Briefly, 20 freshly prepared microcapsules were dried and randomly taken to access the morphological characteristics and microcapsules’ diameter using an optical microscope (Nikon SM2800, Japan mounted with Toup-view Photonics, Co., Ltd Hangzhou, China).
SEM (Neon 40EsB FIB-SEM; Zeiss, Oberkochen, Germany) was used to measure the surface morphology of microcapsules. Multiple pictures from different angles and multiple scales were performed to capture the details of the surface topography. To interpret atoms distribution present in microcapsules, was obtained by using EDS (INCA X-Act; Oxford Instruments, UK). Before analysis, dried microcapsules were mounted on a glass stub and coated under vacuum10 (link).
Briefly, 20 freshly prepared microcapsules were dried and randomly taken to access the morphological characteristics and microcapsules’ diameter using an optical microscope (Nikon SM2800, Japan mounted with Toup-view Photonics, Co., Ltd Hangzhou, China).
SEM (Neon 40EsB FIB-SEM; Zeiss, Oberkochen, Germany) was used to measure the surface morphology of microcapsules. Multiple pictures from different angles and multiple scales were performed to capture the details of the surface topography. To interpret atoms distribution present in microcapsules, was obtained by using EDS (INCA X-Act; Oxford Instruments, UK). Before analysis, dried microcapsules were mounted on a glass stub and coated under vacuum10 (link).
8
SEM Analysis of V. cholerae Binding
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SEM was done as described [25 (link)]. Mid-log phase cultures of V. cholerae O395 were diluted 1:50 into 2 ml of LB broth containing of ZAC-3 IgG (9 μg/ml) or SyH7 IgG and incubated at 37°C for 60 min with aeration. Samples were captured on 0.2 μm polycarbonate filters using a vacuum apparatus and fixed with 2% glutaraldehyde for 20 min, washed with PBS, sterile water, and then subjected to a series of ethanol dehydrations (5 min each). The samples were critical point dried, mounted on aluminum studs with carbon paste and sputter coated with gold for 45 sec. Samples were imaged on a 179 Zeiss Neon-40 EsB FIB-SEM.
9
Microcapsule Characterization: Extensive Analyses
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Microcapsule morphology was analysed using scanning electron microscopy, chemical stability was analysed using Fourier Transformer Infra-Red spectroscopy, thermal stability was analysed using differential scanning calorimetry, and electrokinetic stability was analysed using Zeta-potential analyser, as per our established systems.18 (link),28 (link) In brief, morphology was analysed using Zeiss Neon 40EsB FIBSEM (USA) of freshly made microcapsules, which were coated and multiple images taken, while for chemical analysis, microcapsules were placed in PerkinElmer Spectrum 2 IR (PerkinElmer, USA) and analysed for chemical bond-formation spectrometry. For thermal analysis, melting points were measured for the different microcapsules using single furnace DSC 6000 instrumentation (PerkinElmer, USA) via isothermal analysis, while for electrokinetic stability, electrokinetic potential of the formulations were assessed using the Malvern Zetasizer 3000HSa (Malvern, UK), using 1mL of microencapsulating formulations for each analysis, as per our well-established methodologies.29 (link)–31 (link)
10
Synthesis of Silica Microparticles
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Silica micro particles are produced by a single growth step Stöber synthesis [47] in a semibatch process (continuous addition). This is considered to be the best method to ensure sphericity and monodispersity [48] (link). We add small amounts of electrolyte (NaCl, Sigma-Aldrich) to the initial ammonia solution, at concentrations ranging from 1 to 4 mM, to achieve better control of the final particle size [49] (link). Particles are separated by centrifugation and then washed several times with ethanol 96% (GPR Rectapur VWR) and with de-ionized water (Aquadem 18 MΩ) to eliminate impurities and solvents. They are finally dried at 60 • C for several days.
The particle sizes in all batches (for both rheometry and bending test) were measured by DLS (Malvern Zetasizer Nano S). In addition, we collected SEM micrographs (Zeiss Neon 40 EsB FIB-SEM) of the particles used in Laser Tweezers experiments to have more accurate size measurements; we found them to present a mean diameter d = (1860 ± 90) nm and 5 % polydispersity, which compares well with the DLS results (d = (1960 ± 430) nm).
PMMA particles were purchased from MicroBeads (Spheromers CA3). Particle sizes are distributed around 3 µm with a 15% polydispersity. The particles were washed several times with ethanol and de-ionized water before use.
The particle sizes in all batches (for both rheometry and bending test) were measured by DLS (Malvern Zetasizer Nano S). In addition, we collected SEM micrographs (Zeiss Neon 40 EsB FIB-SEM) of the particles used in Laser Tweezers experiments to have more accurate size measurements; we found them to present a mean diameter d = (1860 ± 90) nm and 5 % polydispersity, which compares well with the DLS results (d = (1960 ± 430) nm).
PMMA particles were purchased from MicroBeads (Spheromers CA3). Particle sizes are distributed around 3 µm with a 15% polydispersity. The particles were washed several times with ethanol and de-ionized water before use.
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