Gene Expression in Electron-Beam-Irradiated Bacteria in Reply to “Live Cell Electron Microscopy Is Probably Impossible”

Kennedy, Eamonn; Nelson, Edward M.; Damiano, John; Timp, G.

doi:10.1021/acsnano.6b06616

Cited by 22 publications

(21 citation statements)

References 25 publications

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“…57 Previous works have shown that the effects of changes in pressure, temperature, free radical generation, and exposure to the electron beam itself are non-significant under low electron doses for large samples, such as those containing E. coli, as implemented here (SI notes). 57,59,61,[65][66][67][68][69][70][71][72][73][74][75][76] Thus, previous works indicate that E. coli 1) maintain their metabolic processes, 2) maintain their cell envelopes, and 3) continue to undergo binary fission within liquid-phase TEM. 57,59,61,[65][66][67][68][69][70][71][72][73][74][75][76] Liquid-phase TEM (Figures 3 and S2) provides further evidence of the penetration of nanoprotrusions in E.coli, as also observed forensically with conventional TEM (Figures 2 and S1).…”

Section: Cell Wallmentioning

confidence: 99%

<p>Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology</p>

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Firlar

Jakubonis

et al. 2020

IJN

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Background: Nanoscale surface roughness has been suggested to have antibacterial and antifouling properties. Several existing models have attempted to explain the antibacterial mechanism of nanoscale rough surfaces without direct observation. Here, conventional and liquid-cell TEM are implemented to observe nanoscale bacteria/surface roughness interaction. The visualization of such interactions enables the inference of possible antibacterial mechanisms. Methods and Results: Nanotextures are synthesized on biocompatible polymer microparticles (MPs) via plasma etching. Both conventional and liquid-phase transmission electron microscopy observations suggest that these MPs may cause cell lysis via bacterial binding to a single protrusion of the nanotexture. The bacterium/protrusion interaction locally compromises the cell wall, thus causing bacterial death. This study suggests that local mechanical damage and leakage of the cytosol kill the bacteria first, with subsequent degradation of the cell envelope. Conclusion: Nanoscale surface roughness may act via a penetrative bactericidal mechanism. This insight suggests that future research may focus on optimizing bacterial binding to individual nanoscale projections in addition to stretching bacteria between nanopillars. Further, antibacterial nanotextures may find use in novel applications employing particles in addition to nanotextures on fibers or films.

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Section: Cell Wallmentioning

confidence: 99%

<p>Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology</p>

Banner

Firlar

Jakubonis

et al. 2020

IJN

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“…64 The dose values are estimates based on experiments performed by Hitachi HT7700. For STEM imaging, the electron dose was kept lower than the critical dose reported by Kennedy et al 65 During TEM imaging, both high and low magnification imaging were carried out on most of the cells on the grids, with the main aim of keeping the cells intact in the vacuum environment.…”

Section: Glc Sample Preparationmentioning

confidence: 99%

“…The observed sequential exocytosis here also matches with the relatively lower resolution video recorded by two-photon excitation microscopy by Takahashi et al 73 The observed full fusion and sequential exocytosis are similar with respect to the ones reported by Takahashi and Kasai 7 and Takahashi et al 73 The EDS characterization in conjunction to similar particle size also confirmed that the particles released are insulin. Kennedy et al 65 was able to monitor the cellular functions by the expression of green fluorescing protein-GFP-LVA by Escherichia coli in the electron microscope. We used an electron dose that is tolerant for the live cell imaging.…”

Section: Fusion and Exocytosis Eventsmentioning

confidence: 99%

“…Furthermore, graphene is 1) impermeable to ion flow, so the liquid medium cannot leak from the graphene cell, and thus prevents the cell drying; 2) electron transparent, so electrons do not accumulate on the sample, preventing electron charging induced damage; 3) thin (~1-2 nm) due to its monolayer nature compared to thicker Si 3 N 4 windows (~30-100 nm) used in earlier works, therefore, formation of secondary electrons is in a lower extent, thus less radiation damage; and 4) a radical scavenger, continuously removing the radiolysis by-products. In addition to these advantageous features of graphene, the electron doses used in this work are within the limits of live cell imaging, and in addition to the cell viability test with fluorescence imaging, similar to the experimentation by Kennedy et al, 65 cellular function specific to the β-cell under consideration, that is insulin secretion, was monitored. 65 Furthermore, the authors did not observe insulin secretion in many of the cells they monitored.…”

Section: Fusion and Exocytosis Eventsmentioning

confidence: 99%

“…In addition to these advantageous features of graphene, the electron doses used in this work are within the limits of live cell imaging, and in addition to the cell viability test with fluorescence imaging, similar to the experimentation by Kennedy et al, 65 cellular function specific to the β-cell under consideration, that is insulin secretion, was monitored. 65 Furthermore, the authors did not observe insulin secretion in many of the cells they monitored. Reasons for this could be either the particular cell under observation is not viable or the local glucose concentration in the given graphene pocket is not enough for the initiation of the insulin secretion.…”

Section: Fusion and Exocytosis Eventsmentioning

confidence: 99%

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In situ graphene liquid cell-transmission electron microscopy study of insulin secretion in pancreatic islet cells

Firlar

Ouy

Covnot

et al. 2019

IJN

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BackgroundIslet cell transplantation is one of the key treatments for type 1 diabetes. Understanding the mechanisms of insulin fusion and exocytosis are of utmost importance for the improvement of the current islet cell transplantation and treatment of diabetes. These phenomena have not been fully evaluated due either to the lack of proper dynamic imaging, or the lack of proper cell preservation during imaging at nanoscales.MethodsBy maintaining the native environment of pancreatic β-cells between two graphene monolayer sheets, we were able to monitor the subcellular events using in situ graphene liquid cell (GLC)-transmission electron microscopy (TEM) with both high temporal and high spatial resolution.ResultsFor the first time, the nucleation and growth of insulin particles until the later stages of fusion were imaged at nanometer scales. The release of insulin from plasma membrane involves the degradation of plasma membrane and drastic reductions in the shorter axis of the insulin particles. Sequential exocytosis results indicated the nucleation, growth and attachment of the new insulin particles to the already anchored ones, which is thermodynamically favorable due to the reduction in total surface, further reducing the Gibbs free energy. The retraction of the already anchored insulin toward the cell is also monitored for the first time live at nanoscale resolution.ConclusionInvestigation of insulin granule dynamics in β-cells can be investigated via GLC-TEM. Our findings with this technology open new realms for the development of novel drugs on pathological pancreatic β-cells, because this approach facilitates observing the effects of the stimuli on the live cells and insulin granules.

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Electron Microscopy Imaging Applications of Room Temperature Ionic Liquids in the Biological Field: A Review

et al. 2021

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For biological imaging using electron microscopy (EM), the use of room‐temperature ionic liquids (RTILs) has been proposed as an alternative to traditional lengthy preparation methods. With their low vapor pressures and conductivity, RTILs can be applied onto hard‐to‐image soft and/or wet samples without dehydration – allowing for a more representative, hydrated state of material and opening the possibility for visualization of in situ physiological processes using conventional EM systems. However, RTILs have yet to be utilized to their full potential by microscopists and microbiologists alike. To this end, this review aims to provide a comprehensive summary of biological applications of RTILs for EM to bridge the RTIL, in situ microscopy, and biological communities. We outline future research avenues for the use of RTILs for the EM observation of biological samples, notably i) RTIL selection and optimization, ii) applications for live cell processes and iii) electron beam and ionic liquid interaction studies.

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Gene Expression in Electron-Beam-Irradiated Bacteria in Reply to “Live Cell Electron Microscopy Is Probably Impossible”

Cited by 22 publications

References 25 publications

<p>Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology</p>

<p>Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology</p>

In situ graphene liquid cell-transmission electron microscopy study of insulin secretion in pancreatic islet cells

Electron Microscopy Imaging Applications of Room Temperature Ionic Liquids in the Biological Field: A Review

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