The Experts below are selected from a list of 14688 Experts worldwide ranked by ideXlab platform
Montserrat Barcena - One of the best experts on this subject based on the ideXlab platform.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
Journal of Structural Biology, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Cryo-focused ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be hindered by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with crinkling of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from such forces, we milled "micro-expansion joints" alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of micro-expansion joints drastically decreases bending of Lamellae milled from eukaryotic cells grown and frozen on EM grids. Furthermore, we show that this adaptation does not create additional instabilities that could impede subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them a straightforward solution against cryo-Lamella bending to increase the throughput of in situ structural biology studies.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
bioRxiv, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Abstract Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from these forces, we milled “micro-expansion joints” alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases Lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-Lamella bending in any biological sample milled on EM grids.
Georg Wolff - One of the best experts on this subject based on the ideXlab platform.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
Journal of Structural Biology, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Cryo-focused ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be hindered by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with crinkling of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from such forces, we milled "micro-expansion joints" alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of micro-expansion joints drastically decreases bending of Lamellae milled from eukaryotic cells grown and frozen on EM grids. Furthermore, we show that this adaptation does not create additional instabilities that could impede subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them a straightforward solution against cryo-Lamella bending to increase the throughput of in situ structural biology studies.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
bioRxiv, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Abstract Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from these forces, we milled “micro-expansion joints” alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases Lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-Lamella bending in any biological sample milled on EM grids.
Kohji Tashiro - One of the best experts on this subject based on the ideXlab platform.
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structural changes in isothermal crystallization process of polyoxymethylene investigated by time resolved ftir saxs and waxs measurements
Polymer, 2003Co-Authors: Hisakatsu Hama, Kohji TashiroAbstract:Abstract Structural evolution in the isothermal crystallization process of polyoxymethylene from the molten state has been investigated by carrying out the time-resolved measurements of infrared spectra and synchrotron small angle X-ray scattering (SAXS) and wide angle X-ray scattering. In case of isothermal crystallization at 130 °C, for example, the infrared bands intrinsic of folded chain crystal (FCC) morphology appeared at first, and then the bands of extended chain crystal (ECC) morphology were detected with time delay of ca. 150 s. In the SAXS experiment at 130 °C, the Lamellar stacking structure of the long period of ca. 15 nm was observed at first, which changed rapidly to ca. 12 nm in a short time. The SAXS peak with the long period of ca. 6 nm started to appear with a time delay of ca. 150 s after the initial Lamellae appeared and coexisted with the initially-observed 12 nm peak. Judging from the timing to detect these characteristic infrared and SAXS signals, a good correspondence was found to exist between the stacked Lamellar structure of 12 nm long period and FCC morphology and between the structure of 6 nm long period and ECC morphology. The quantitative analysis was made for the SAXS data on the basis of the Lamellar insertion model combined with the paracrystalline theory of the second-kind of disorder. The following structural evolution was deduced from all these results. Immediately after the temperature jump from the melt to 130 °C, the stacked Lamellar structure of FCC morphology was generated at first. New Lamellae were formed from the amorphous region in between the originally-existing Lamellae about 150 s later, where the random chain segments bridging the adjacent Lamellae were extended to form the taut tie chains, giving infrared bands of ECC morphology. This inserted Lamellar structure of 6 nm long period coexisted at a population of ca. 6% with the initially-formed Lamellar stacking structure of 12 nm long period. When the experiment was made at 150 °C, only the formation of stacked Lamellar structure of FCC morphology was observed and the insertion of new Lamella did not occur.
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structural changes in non isothermal crystallization process of melt cooled polyoxymethylene i detection of infrared bands characteristic of folded and extended chain crystal morphologies and extraction of a Lamellar stacking model
Polymer, 2003Co-Authors: Hisakatsu Hama, Kohji TashiroAbstract:Abstract Structural change in the crystallization process of polyoxymethylene (POM) cooled from the molten state has been investigated by the measurements of infrared spectra and small-angle (SAXS) and wide-angle X-ray scatterings (WAXS). When the melt was cooled slowly, the infrared bands characteristic of a folded chain crystal (FCC) were observed to appear around 156 °C. Below 140 °C, the infrared bands intrinsic of an extended chain crystal (ECC) were detected to increase in intensity. In the SAXS measurement, the peak ( L 1 ) corresponding to a stacked Lamellar structure with the long period of ca. 14 nm was found to grow in parallel to the growth of infrared FCC bands. In the temperature region of the observation of infrared ECC bands, the new peak ( L 2 ) of long period of ca. 7 nm was found to appear and the intensity exchange occurred between the L 1 and L 2 peaks, that is, with decreasing temperature the L 2 peak increased the intensity and its height became comparable to the L 1 peak height. By combining all these experimental data, a model to illustrate the formation process of Lamellar stacking structure has been presented. After the appearance of stacked Lamellar structure of 14 nm long period from the melt, new Lamellae are created in between the already existing Lamellae and the long period changes to the half value, 7 nm. Some of molecular chain stems in a Lamella are speculated to pass through the adjacent Lamellae to form a bundle of fully extended taut tie chains, which are considered to be observed as the infrared bands characteristic of ECC morphology. Although the POM samples used in this experiment may contain small amount of low-molecular-weight macrocyclic component, it was not plausible judging from the various experimental data to assign the secondarily observed 7 nm SAXS peak to the repeating period originating from the stacked structure of macrocyclic compounds.
David A Agard - One of the best experts on this subject based on the ideXlab platform.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
Journal of Structural Biology, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Cryo-focused ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be hindered by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with crinkling of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from such forces, we milled "micro-expansion joints" alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of micro-expansion joints drastically decreases bending of Lamellae milled from eukaryotic cells grown and frozen on EM grids. Furthermore, we show that this adaptation does not create additional instabilities that could impede subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them a straightforward solution against cryo-Lamella bending to increase the throughput of in situ structural biology studies.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
bioRxiv, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Abstract Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from these forces, we milled “micro-expansion joints” alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases Lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-Lamella bending in any biological sample milled on EM grids.
Eric J Snijder - One of the best experts on this subject based on the ideXlab platform.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
Journal of Structural Biology, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Cryo-focused ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be hindered by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with crinkling of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from such forces, we milled "micro-expansion joints" alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of micro-expansion joints drastically decreases bending of Lamellae milled from eukaryotic cells grown and frozen on EM grids. Furthermore, we show that this adaptation does not create additional instabilities that could impede subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them a straightforward solution against cryo-Lamella bending to increase the throughput of in situ structural biology studies.
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mind the gap micro expansion joints drastically decrease the bending of fib milled cryo Lamellae
bioRxiv, 2019Co-Authors: Georg Wolff, Ronald W A L Limpens, Shawn Q Zheng, Eric J Snijder, David A Agard, Abraham J Koster, Montserrat BarcenaAbstract:Abstract Cryo-focussed ion beam (FIB)-milling of biological samples can be used to generate thin electron-transparent slices from cells grown or deposited on EM grids. These so called cryo-Lamellae allow high-resolution structural studies of the natural cellular environment by in situ cryo-electron tomography. However, the cryo-Lamella workflow is a low-throughput technique and can easily be obstructed by technical issues like the bending of the Lamellae during the final cryo-FIB-milling steps. The severity of Lamella bending seems to correlate with shrinkage of the EM grid support film at cryogenic temperatures, which could generate tensions that may be transferred onto the thin Lamella, leading to its bending and breakage. To protect the Lamellae from these forces, we milled “micro-expansion joints” alongside the Lamellae, creating gaps in the support that can act as physical buffers to safely absorb material motion. We demonstrate that the presence of such micro-expansion joints drastically decreases Lamella bending. Furthermore, we show that this adaptation does not create instabilities that could constrain subsequent parts of the cryo-Lamella workflow, as we obtained high-quality Volta phase plate tomograms revealing macromolecules in their natural structural context. The minimal additional effort required to implement micro-expansion joints in the cryo-FIB-milling workflow makes them an easy solution against cryo-Lamella bending in any biological sample milled on EM grids.
