TEM was used to visualise the ultrastructure of the pellicle in selected enamel samples from one subject. After an oral exposure of 8 h, the samples were fixed using a glutaraldehyde solution (2.5% glutaraldehyde, 1.5% formaldehyde in phosphate buffer). After osmication with 1% osmium tetroxide stock solution and 0.1 M cacodylate buffer, the enamel samples were dehydrated in an ascending ethanol series diluted in distilled water (50%, 70%, 90%, 100%), 100% acetone and an Araldite-acetone mixture (1:1), followed by embedding in an Araldite mixture (Plano, Wetzlar, Germany). Remaining dentin on the samples was removed using 1200-grit sandpaper. The enamel samples were decalcified in 1 M HCl and counter-embedded in an Araldite mixture. Ultrathin sections were made using Reichert Ultracut E (FEI Eindhoven, The Netherlands). If necessary, the samples were post-stained with uranyl acetate and lead citrate. TEM investigations were conducted using TEM TECNAI 12 Biotwin (FEI Eindhoven, The Netherlands).
Tecnai 12 biotwin tem
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands, United States
The Tecnai 12 Biotwin TEM is a transmission electron microscope (TEM) designed for biological specimen analysis. It provides high-resolution imaging capabilities for the examination of cellular and molecular structures. The Tecnai 12 Biotwin TEM is a tool for researchers and scientists working in the fields of biology, biochemistry, and materials science.
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Lab products found in correlation
Renlam m 1 resin, by Serva Electrophoresis (2 mentions)
Em uc7, by Leica camera (1 mentions)
Leica em uc7, by Leica Microsystems (1 mentions)
Jsm 7600f sem, by JEOL (1 mentions)
Araldite m, by Serva Electrophoresis (1 mentions)
Carbon coated copper grid, by Electron Microscopy Sciences (1 mentions)
Leica ultracut uct ultramicrotome, by Leica Microsystems (1 mentions)
Rbma 200c, by World Precision Instruments (1 mentions)
Filter paper, by Cytiva (1 mentions)
Lacy formvar carbon film, by ProSciTech (1 mentions)
Fei eagle 4k 4k ccd, by Thermo Fisher Scientific (1 mentions)
Diamond knife, by Electron Microscopy Sciences (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Megaview 3 wide angle camera, by EMSIS (1 mentions)
Epon 812 resin, by SPI Supplies (1 mentions)
Araldite cy212, by Agar Scientific (1 mentions)
Empact2, by Leica Microsystems (1 mentions)
Lowicryl hm20 resin, by Polysciences (1 mentions)
Morgagni tem, by Thermo Fisher Scientific (1 mentions)
Gs 6207, by Gilead Sciences (1 mentions)
27 protocols using tecnai 12 biotwin tem
1
Ultrastructural Analysis of Enamel Pellicle
2
TEM Analysis of Biofilm Ultrastructure
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TEM analysis was performed on specimens carried by two volunteers in order to visualize the ultrastructure of the biofilm. After removal of the splints, all the four different samples followed a rigorous protocol according to the methods in Nobre et al. [19 (link)]. Except that in the present experiment, the following additional procedure was performed only for titanium and ceramics samples: after polymerization, titanium and ceramics were removed by treatment with hydrofluoric acid (5%) during 48 h, and the specimens were re-embedded in Araldite. The enamel was decalcified due to exposure in 0.1M HCl for 4 h, and the specimens were re-embedded in Araldite.
As the final step, the specimens were cut in ultra-thin sections in an ultramicrotome with a diamond knife (Leica EM UC7, Germany) and mounted on Pioloform-coated copper grids and contrasted with aqueous solutions of uranyl acetate and lead citrate at room temperature. After intensive washing with distilled water, biofilms were analyzed with a TEM Tecnai 12 Biotwin (FEI, Eindhoven, The Netherlands) under a magnification up to 100,000-fold.
As the final step, the specimens were cut in ultra-thin sections in an ultramicrotome with a diamond knife (Leica EM UC7, Germany) and mounted on Pioloform-coated copper grids and contrasted with aqueous solutions of uranyl acetate and lead citrate at room temperature. After intensive washing with distilled water, biofilms were analyzed with a TEM Tecnai 12 Biotwin (FEI, Eindhoven, The Netherlands) under a magnification up to 100,000-fold.
3
Ultrastructural Analysis of Dental Pellicle
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The pellicle-covered enamel slabs were fixed in 2.5% glutaraldehyde/1.5% formaldehyde for 2 h. Postfixation took place in 1% osmium tetroxide for 2 h. The specimens were dehydrated in an ascending series of alcohol and embedded in Araldite CY 212 (Plano, Wetzlar, Germany). After decalcification in 1 M HCl re-embedding was accomplished with Araldite. Ultrathin sections of the pellicle layer were cut with a diamond knife, mounted on pioloform-coated copper grids and contrasted with uranyl acetate and lead citrate. TEM analysis of the pellicle´s ultrastructure was performed at 30,000–100,000 fold magnification in a TEM TECNAI 12 Biotwin (FEI Eindhoven, The Netherlands).
4
Transmission Electron Microscopy of Gradient Samples
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After 8 h of oral exposure, gradient specimens were processed for TEM analysis. After fixing in 1% glutaraldehyde/1% paraformaldehyde for 2 h, specimens were post-fixed in 2% osmium tetroxide. Dehydration took place in an ascending series of ethanol. Specimens were embedded in Araldite CY212 (Agrar Scientific, Stansted, UK) and ultrathin sections were cut using a diamond knife. The ultrathin sections were contrasted with uranyl acetate and lead citrate and analyzed in a TEM TECNAI 12 Biotwin (FEI, Eindhoven, The Netherlands). Representative micrographs were taken at magnifications of 1000-fold up to 90,000 fold.
5
Visualizing Bacteria-Substance Interactions
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In order to visualize the interaction of test substances with bacteria, the saliva of two subjects was additionally examined with TEM. The subjects rinsed for 30 s with 10 mL of a test substance and their unstimulated saliva was collected after 1 min, 30 min and 2 h in an Eppendorf tube. The samples were centrifuged at 5000 rpm and the bacterial pellet was fixed in a fixing solution consisting of 1% formaldehyde, 1% glutaraldehyde and 0.1 M cacodylate buffer for 90 min. Then, samples were postfixed with 2% osmium for 1 h and pre-embedded in low-melting agarose. After dehydration in an ascending alcohol series, samples were embedded in araldite (Araldit CY212, Agar Scientific Ltd., Stansted, UK). Ultrathin sections of the embedded samples were cut in an ultramicrotome (Leica EM UC7, Leica Microsystems, Wetzlar, Germany). The sections were contrasted with UranyLess (UranyLess EM Stain, Delta Microscopies, Mauressac, France) and 3% lead citrate before investigated by transmission electron microscopy (TEM Tecnai 12 BioTwin, FEI Company, Eindhoven, Netherlands) at magnifications of up to 68,000-fold.
6
Ultrastructural Analysis of Eroded Dental Specimens
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After in vitro erosion or after specimens have been removed from the oral cavity, they were rinsed with sterile water and fixed with glutaraldehyde for 2 h. Then, specimens were washed in phosphate buffer, postfixed in 2% osmium tetroxide for 2 h, and washed in phosphate buffer again. The specimens were embedded in Araldite CY 212 (Serva, Heidelberg, Germany), and ultrathin sections were cut using an Ultracut E ultra-microtome (Reichert, Bensheim, Germany). Finally, the samples were stained with uranyl acetate and lead citrate and analyzed with a transmission electron microscope (TEM Tecnai 12 BioTwin, FEI Co., Eindhoven, The Netherlands) at a magnification of up to 100,000-fold.
7
Synthesis and Characterization of Silver Nanoparticles in Cellulose
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The synthesis of silver nanoparticles in the cellulose was observed by UV-Vis spectrophotometry (from 300–600 nm), scanning electron microscopy (SEM), and transmission electron microscopy (TEM)36 . For SEM, samples were dried and covered with gold/palladium (around 4 nm thickness) then analyzed on a JEOL JSM-7600F SEM. TEM images of cellulose were acquired using a TEM Tecnai 12 BioTWIN (manufactured by FEI) coupled to a SIS Megaview 3 camera at acceleration voltage of 120 kV. Before the analysis, diluted samples (0.2% aqueous) were sonicated for 30 min using an ice-cold ultrasound bath (output of 1200 W). Drops of the sample (about 8 μL) were left on the grid for 5 min, then negatively stained with 1% uranyl acetate and finally glow discharged. Copper grids with a formvar/carbon support film were used.
8
Ultrastructural Analysis of Dental Pellicle
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After 30 min and 2 h of intraoral exposure pellicle-covered enamel slabs were fixed in 2.5% glutaraldehyde/1.5% formaldehyde for 2 h. Postfixation took place in 1% osmium tetroxide for 2 h. The specimens were dehydrated in an ascending series of alcohol and embedded in Araldite CY 212 (Plano, Wetzlar, Germany). After decalcification in 1 M HCl, reembedding was accomplished with Araldite. Ultrathin sections of the pellicle layer were cut as described above, mounted on pioloform-coated copper grids and contrasted with uranyl acetate and lead citrate. TEM analysis of the pellicle's ultrastructure was performed at 30,000-to 100,000-fold magnification in a TEM TECNAI 12 Biotwin (FEI, Eindhoven, The Netherlands).
9
Visualizing Pellicle Ultrastructure
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Furthermore, in situ pellicle samples were gained for TEM analysis in order to visualize the influence of the plant extracts on the ultrastructure of the pellicle. The in situ experiments were carried out as described above. Some of the samples were incubated in HCl for 60 s after the intraoral exposure. Afterwards, the slabs were transferred to preparation for TEM. In a first step, the enamel slabs were fixed in glutaraldehyde for 2 h (2.5% glutaraldehyde, 1.5% formaldehyde in phosphate buffer, pH 7.4). The samples were then washed 5 times in phosphate buffer. Postfixation for visualization of organic structures took place in 1% osmium tetroxide for 2 h. The specimens were dehydrated in a series of increasing alcohol concentrations and embedded in Araldite M (Serva, Darmstadt, Germany). The dentine was removed from the samples with a diamond bur and the samples were decalcified in 1 M HCl. Re-embedding was performed with Araldite. Ultrathin sections of the pellicle samples were cut in series with an ultramicrotome (Ultracut E, Reichert, Bensheim, Germany) using a diamond knife. The ultrathin sections were mounted on mesh grids (Plano, Wetzlar, Germany) and contrasted with uranyl acetate and lead citrate. TEM investigation took place at 3,000-to 50,000-fold magnification in a TEM TECNAI 12 Biotwin (FEI, Eindhoven, The Netherlands) [Hannig et al., 2012] .
10
Negative Staining of Hydrogel Samples
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Hydrogels were
first diluted 10-fold using ddH2O and then negatively stained.
A carbon-coated copper grid (400 mesh from Electron Microscopy Sciences)
was placed on a 10 μL droplet of sample for 1 min and excess
liquid drained off using lint-free tissue. The grid was then placed
on a 10 μL droplet of ddH2O for 10 s before excess
liquid was drained off. The grid was then transferred to a 10 μL
droplet of a 1% uranyl acetate solution for 30 s, and again excess
liquid was drained off. Finally, the grid was transferred to a 10
μL droplet of ddH2O for 10 s before excess liquid
was drained off for a final time. The sample was then left to dry
before imaging using a FEI Tecnai12 BioTwin TEM at 100 keV.
first diluted 10-fold using ddH2O and then negatively stained.
A carbon-coated copper grid (400 mesh from Electron Microscopy Sciences)
was placed on a 10 μL droplet of sample for 1 min and excess
liquid drained off using lint-free tissue. The grid was then placed
on a 10 μL droplet of ddH2O for 10 s before excess
liquid was drained off. The grid was then transferred to a 10 μL
droplet of a 1% uranyl acetate solution for 30 s, and again excess
liquid was drained off. Finally, the grid was transferred to a 10
μL droplet of ddH2O for 10 s before excess liquid
was drained off for a final time. The sample was then left to dry
before imaging using a FEI Tecnai12 BioTwin TEM at 100 keV.
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