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The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane
Kamischke C, Fan J, Bergeron J, Kulasekara HD, Dalebroux ZD, Burrell A, Kollman JM, Miller SI
eLife 2019;8:e40171 DOI: 10.7554/eLife.40171
The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumanniitransposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.
Kamischke C, Fan J, Bergeron J, Kulasekara HD, Dalebroux ZD, Burrell A, Kollman JM, Miller SI
eLife 2019;8:e40171 DOI: 10.7554/eLife.40171
The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumanniitransposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.
A Polysulfide-Triggered Fluorescent Indicator Suitable for Super-Resolution Microscopy and Application in Imaging
Hoskere A , Sreedharan S, Ali F, Smythe CG, Thomas JA and Das A,
Chem Commun, 2018, 54, 3735-373
We have developed an organic molecule which could image the in-cellulo generated reactive species like sulfide which are produced due to the biological processes happening in cells through different signalling mechanism involving vital sub-cellular organelles like Endoplasmic reticulum. This organic molecule functions as a super resolution microscopy probe capturing the signalling mechanism happening by trapping the sulfide species. This information could be useful to develop therapeutics for cardiovascular disorders.
Hoskere A , Sreedharan S, Ali F, Smythe CG, Thomas JA and Das A,
Chem Commun, 2018, 54, 3735-373
We have developed an organic molecule which could image the in-cellulo generated reactive species like sulfide which are produced due to the biological processes happening in cells through different signalling mechanism involving vital sub-cellular organelles like Endoplasmic reticulum. This organic molecule functions as a super resolution microscopy probe capturing the signalling mechanism happening by trapping the sulfide species. This information could be useful to develop therapeutics for cardiovascular disorders.
Mitochondria-localising DNA-binding biscyclometalated phenyltriazole iridium(III) dipyridophenazene complexes: syntheses and cellular imaging properties
Sreedharan S, Sinopoli A, Jarman P, Robinson D, Clemmet C, Scattergood PA, Rice CR, Smythe CGW, Thomas JA and Elliott PIP.
Dalton Trans, 2018, 47, 4931–4940.
Functionalised metal complexes are molecules which can bind to DNA and they function as imaging agents/photodynamic therapeutics, here in this paper we showed a metal complex with luminescent properties which could bind to DNA could indeed be taken-up by mitochondria and hence could image them and could potentially be optimised for drug delivery application specifically to mitochondria.
Sreedharan S, Sinopoli A, Jarman P, Robinson D, Clemmet C, Scattergood PA, Rice CR, Smythe CGW, Thomas JA and Elliott PIP.
Dalton Trans, 2018, 47, 4931–4940.
Functionalised metal complexes are molecules which can bind to DNA and they function as imaging agents/photodynamic therapeutics, here in this paper we showed a metal complex with luminescent properties which could bind to DNA could indeed be taken-up by mitochondria and hence could image them and could potentially be optimised for drug delivery application specifically to mitochondria.
High-speed large area atomic force microscopy using a quartz resonator.
Wang JY, Mullin N, Hobbs J.
Nanotechnology. 2018 May 24. doi: 10.1088/1361-6528/aac7a3. [Epub ahead of print]
A high-speed atomic force microscope for scanning large areas, utilising a quartz bar driven close to resonance to provide the motion in the fast scan axis is presented. Images up to 170 x 170 μm2 have been obtained on a polydimethylsiloxane (PDMS) grating in 1 second. This is provided through an average tip-sample velocity of 28 cm/sec at a line rate of 830 Hz. Scan areas up to 80 x 80 μm2 have been obtained in 0.42 seconds with a line rate of 1410 Hz. To demonstrate the capability of the scanner the spherulitic crystallization of a semicrystalline polymer was imaged in situ at high speed.
Wang JY, Mullin N, Hobbs J.
Nanotechnology. 2018 May 24. doi: 10.1088/1361-6528/aac7a3. [Epub ahead of print]
A high-speed atomic force microscope for scanning large areas, utilising a quartz bar driven close to resonance to provide the motion in the fast scan axis is presented. Images up to 170 x 170 μm2 have been obtained on a polydimethylsiloxane (PDMS) grating in 1 second. This is provided through an average tip-sample velocity of 28 cm/sec at a line rate of 830 Hz. Scan areas up to 80 x 80 μm2 have been obtained in 0.42 seconds with a line rate of 1410 Hz. To demonstrate the capability of the scanner the spherulitic crystallization of a semicrystalline polymer was imaged in situ at high speed.
Molecular imaging of glycan chains couples cell-wall polysaccharide architecture to bacterial cell morphology.
Turner RD, Mesnage S, Hobbs JK, Foster SJ.
Nat Commun. 2018 Mar 28;9(1):1263.
Biopolymer composite cell walls maintain cell shape and resist forces in plants, fungi and bacteria. Peptidoglycan, a crucial antibiotic target and immunomodulator, performs this role in bacteria. The textbook structural model of peptidoglycan is a highly ordered, crystalline material. Here we use atomic force microscopy (AFM) to image individual glycan chains in peptidoglycan from Escherichia coli in unprecedented detail. We quantify and map the extent to which chains are oriented in a similar direction (orientational order), showing it is much less ordered than previously depicted. Combining AFM with size exclusion chromatography, we reveal glycan chains up to 200 nm long. We show that altered cell shape is associated with substantial changes in peptidoglycan biophysical properties. Glycans from E. coli in its normal rod shape are long and circumferentially oriented, but when a spheroid shape is induced (chemically or genetically) glycans become short and disordered.
Turner RD, Mesnage S, Hobbs JK, Foster SJ.
Nat Commun. 2018 Mar 28;9(1):1263.
Biopolymer composite cell walls maintain cell shape and resist forces in plants, fungi and bacteria. Peptidoglycan, a crucial antibiotic target and immunomodulator, performs this role in bacteria. The textbook structural model of peptidoglycan is a highly ordered, crystalline material. Here we use atomic force microscopy (AFM) to image individual glycan chains in peptidoglycan from Escherichia coli in unprecedented detail. We quantify and map the extent to which chains are oriented in a similar direction (orientational order), showing it is much less ordered than previously depicted. Combining AFM with size exclusion chromatography, we reveal glycan chains up to 200 nm long. We show that altered cell shape is associated with substantial changes in peptidoglycan biophysical properties. Glycans from E. coli in its normal rod shape are long and circumferentially oriented, but when a spheroid shape is induced (chemically or genetically) glycans become short and disordered.
Dynamic thylakoid stacking regulates the balance between linear and cyclic photosynthetic electron transfer.
Wood WHJ, MacGregor-Chatwin C, Barnett SFH, Mayneord GE, Huang X, Hobbs JK, Hunter CN, Johnson MP.
Nat Plants. 2018 Feb;4(2):116-127.
Upon transition of plants from darkness to light the initiation of photosynthetic linear electron transfer (LET) from H2O to NADP+ precedes the activation of CO2 fixation, creating a lag period where cyclic electron transfer (CET) around photosystem I (PSI) has an important protective role. CET generates ΔpH without net reduced NADPH formation, preventing overreduction of PSI via regulation of the cytochrome b 6 f (cytb 6 f) complex and protecting PSII from overexcitation by inducing non-photochemical quenching. The dark-to-light transition also provokes increased phosphorylation of light-harvesting complex II (LHCII). However, the relationship between LHCII phosphorylation and regulation of the LET/CET balance is not understood. Here, we show that the dark-to-light changes in LHCII phosphorylation profoundly alter thylakoid membrane architecture and the macromolecular organization of the photosynthetic complexes, without significantly affecting the antenna size of either photosystem. The grana diameter and number of membrane layers per grana are decreased in the light while the number of grana per chloroplast is increased, creating a larger contact area between grana and stromal lamellae. We show that these changes in thylakoid stacking regulate the balance between LET and CET pathways. Smaller grana promote more efficient LET by reducing the diffusion distance for the mobile electron carriers plastoquinone and plastocyanin, whereas larger grana enhance the partition of the granal and stromal lamellae plastoquinone pools, enhancing the efficiency of CET and thus photoprotection by non-photochemical quenching.
Wood WHJ, MacGregor-Chatwin C, Barnett SFH, Mayneord GE, Huang X, Hobbs JK, Hunter CN, Johnson MP.
Nat Plants. 2018 Feb;4(2):116-127.
Upon transition of plants from darkness to light the initiation of photosynthetic linear electron transfer (LET) from H2O to NADP+ precedes the activation of CO2 fixation, creating a lag period where cyclic electron transfer (CET) around photosystem I (PSI) has an important protective role. CET generates ΔpH without net reduced NADPH formation, preventing overreduction of PSI via regulation of the cytochrome b 6 f (cytb 6 f) complex and protecting PSII from overexcitation by inducing non-photochemical quenching. The dark-to-light transition also provokes increased phosphorylation of light-harvesting complex II (LHCII). However, the relationship between LHCII phosphorylation and regulation of the LET/CET balance is not understood. Here, we show that the dark-to-light changes in LHCII phosphorylation profoundly alter thylakoid membrane architecture and the macromolecular organization of the photosynthetic complexes, without significantly affecting the antenna size of either photosystem. The grana diameter and number of membrane layers per grana are decreased in the light while the number of grana per chloroplast is increased, creating a larger contact area between grana and stromal lamellae. We show that these changes in thylakoid stacking regulate the balance between LET and CET pathways. Smaller grana promote more efficient LET by reducing the diffusion distance for the mobile electron carriers plastoquinone and plastocyanin, whereas larger grana enhance the partition of the granal and stromal lamellae plastoquinone pools, enhancing the efficiency of CET and thus photoprotection by non-photochemical quenching.
Tracking HOCl concentrations across cellular organelles in real time using a super resolution microscopy probe.
Ali F, Aute S, Sreedharan S, Anila HA, Saeed HK, Smythe CG, Thomas JA, Das A.
Chem Commun (Camb). 2018 Jan 31.
BODIPY derivative, SF-1, exclusively shows a fluorescence ON response to HOCl and images endogenously generated HOCl in RAW 264.7 macrophages. Widefield and super resolution structured illumination microscopy images confirm localization in the Golgi complex and lysosomes, and hence specifically detects HOCl generated in these organelles. SF-1 is compatible with 3D-SIM imaging of individual cells.
Ali F, Aute S, Sreedharan S, Anila HA, Saeed HK, Smythe CG, Thomas JA, Das A.
Chem Commun (Camb). 2018 Jan 31.
BODIPY derivative, SF-1, exclusively shows a fluorescence ON response to HOCl and images endogenously generated HOCl in RAW 264.7 macrophages. Widefield and super resolution structured illumination microscopy images confirm localization in the Golgi complex and lysosomes, and hence specifically detects HOCl generated in these organelles. SF-1 is compatible with 3D-SIM imaging of individual cells.
Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search
Lee AJ, Endo M, Hobbs JK, Wälti C
ACS Nano. 2018 Jan 23;12(1):272-278.
Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.
Lee AJ, Endo M, Hobbs JK, Wälti C
ACS Nano. 2018 Jan 23;12(1):272-278.
Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.
Multimodal Probes: Superresolution and Transmission Electron Microscopy Imaging of Mitochondria, and Oxygen Mapping of Cells, Using Small-Molecule Ir(III) Luminescent Complexes.
Shewring JR, Cankut AJ, McKenzie LK, Crowston BJ, Botchway SW, Weinstein JA, Edwards E, Ward MD.
Inorg Chem. 2017 Dec 18;56(24):15259-15270.
We describe an Ir(III)-based small-molecule, multimodal probe for use in both light and electron microscopy. The direct correlation of data between light- and electron-microscopy-based imaging to investigate cellular processes at the ultrastructure level is a current challenge, requiring both dyes that must be brightly emissive for luminescence imaging and scatter electrons to give contrast for electron microscopy, at a single working concentration suitable for both methods. Here we describe the use of Ir(III) complexes as probes that provide excellent image contrast and quality for both luminescence and electron microscopy imaging, at the same working concentration. Significant contrast enhancement of cellular mitochondria was observed in transmission electron microscopy imaging, with and without the use of typical contrast agents. The specificity for cellular mitochondria was also confirmed with MitoTracker using confocal and 3D-structured illumination microscopy. These phosphorescent dyes are part of a very exclusive group of transition-metal complexes that enable imaging beyond the diffraction limit. Triplet excited-state phosphorescence was also utilized to probe the O2 concentration at the mitochondria in vitro, using lifetime mapping techniques.
Shewring JR, Cankut AJ, McKenzie LK, Crowston BJ, Botchway SW, Weinstein JA, Edwards E, Ward MD.
Inorg Chem. 2017 Dec 18;56(24):15259-15270.
We describe an Ir(III)-based small-molecule, multimodal probe for use in both light and electron microscopy. The direct correlation of data between light- and electron-microscopy-based imaging to investigate cellular processes at the ultrastructure level is a current challenge, requiring both dyes that must be brightly emissive for luminescence imaging and scatter electrons to give contrast for electron microscopy, at a single working concentration suitable for both methods. Here we describe the use of Ir(III) complexes as probes that provide excellent image contrast and quality for both luminescence and electron microscopy imaging, at the same working concentration. Significant contrast enhancement of cellular mitochondria was observed in transmission electron microscopy imaging, with and without the use of typical contrast agents. The specificity for cellular mitochondria was also confirmed with MitoTracker using confocal and 3D-structured illumination microscopy. These phosphorescent dyes are part of a very exclusive group of transition-metal complexes that enable imaging beyond the diffraction limit. Triplet excited-state phosphorescence was also utilized to probe the O2 concentration at the mitochondria in vitro, using lifetime mapping techniques.
Repurposing a photosynthetic antenna protein as a super-resolution microscopy label.
Barnett SFH, Hitchcock A, Mandal AK, Vasilev C, Yuen JM, Morby J, Brindley AA, Niedzwiedzki DM, Bryant DA, Cadby AJ, Holten D, Hunter CN.
Sci Rep. 2017 Dec 1;7(1):16807.
Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd 1 ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.
Barnett SFH, Hitchcock A, Mandal AK, Vasilev C, Yuen JM, Morby J, Brindley AA, Niedzwiedzki DM, Bryant DA, Cadby AJ, Holten D, Hunter CN.
Sci Rep. 2017 Dec 1;7(1):16807.
Techniques such as Stochastic Optical Reconstruction Microscopy (STORM) and Structured Illumination Microscopy (SIM) have increased the achievable resolution of optical imaging, but few fluorescent proteins are suitable for super-resolution microscopy, particularly in the far-red and near-infrared emission range. Here we demonstrate the applicability of CpcA, a subunit of the photosynthetic antenna complex in cyanobacteria, for STORM and SIM imaging. The periodicity and width of fabricated nanoarrays of CpcA, with a covalently attached phycoerythrobilin (PEB) or phycocyanobilin (PCB) chromophore, matched the lines in reconstructed STORM images. SIM and STORM reconstructions of Escherichia coli cells harbouring CpcA-labelled cytochrome bd 1 ubiquinol oxidase in the cytoplasmic membrane show that CpcA-PEB and CpcA-PCB are suitable for super-resolution imaging in vivo. The stability, ease of production, small size and brightness of CpcA-PEB and CpcA-PCB demonstrate the potential of this largely unexplored protein family as novel probes for super-resolution microscopy.
Development of targeted STORM for super resolution imaging of biological samples using digital micro-mirror device.
Peedikakkal LV, Steventon V, Furley A, Cadby AJ.
Optics Communications (2017), 404, 18-22
Peedikakkal LV, Steventon V, Furley A, Cadby AJ.
Optics Communications (2017), 404, 18-22
We demonstrate a simple illumination system based on a digital mirror device which allows for fine control over the power and pattern of illumination. We apply this to localization microscopy (LM), specifically stochastic optical reconstruction microscopy (STORM). Using this targeted STORM, we were able to image a selected area of a labelled cell without causing photo-damage to the surrounding areas of the cell.
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Fig. 5. A 3T3 cell is image using STORM, part A shows the sample illuminated under low power. The sample is illuminated under high power using a heart shape pattern, a single frame from the OFF camera is given in part B. After 10 k frames of high illumination the sample was again re-imaged using low power this is given in part C. Finally, part D shows the average intensity from a region of the sample during illumination for an ON region (BLUE) and OFF region (RED), showing that even the OFF regions are illuminated. |
Cooperative RecA clustering: the key to efficient homology searching.
Lee AJ, Sharma R, Hobbs JK, Walti C.
Nucleic Acids Research (2017); 45(20):11743-11751
This paper studies the interaction of RecA nucleoprotein filaments with DNA. RecA is a DNA repair protein, and it is currently unclear how RecA filaments efficiently locate sequence homology across genomic DNA. Here, using atomic force microscopy, we find that the process is highly cooperative, involving an initial rapid association phase, followed by a slower ‘resolution’ phase. AFM provides unique, quantifiable, single molecule insight into the process.
Lee AJ, Sharma R, Hobbs JK, Walti C.
Nucleic Acids Research (2017); 45(20):11743-11751
This paper studies the interaction of RecA nucleoprotein filaments with DNA. RecA is a DNA repair protein, and it is currently unclear how RecA filaments efficiently locate sequence homology across genomic DNA. Here, using atomic force microscopy, we find that the process is highly cooperative, involving an initial rapid association phase, followed by a slower ‘resolution’ phase. AFM provides unique, quantifiable, single molecule insight into the process.
Multimodal Super-resolution Optical Microscopy Using a Transition-Metal-Based Probe Provides Unprecedented Capabilities for Imaging Both Nuclear Chromatin and Mitochondria.
Sreedharan S, Gill MR, Garcia E, Saeed HK, Robinson D, Byrne A, Cadby A, Keyes TE, Smythe C, Pellett P, Bernardino de la Serna J, Thomas JA.
J Am Chem Soc. (2017) 139(44):15907-15913.
Sreedharan S, Gill MR, Garcia E, Saeed HK, Robinson D, Byrne A, Cadby A, Keyes TE, Smythe C, Pellett P, Bernardino de la Serna J, Thomas JA.
J Am Chem Soc. (2017) 139(44):15907-15913.
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Detailed studies on the live cell uptake properties of a dinuclear membrane-permeable RuII cell probe show that, at low concentrations, the complex localizes and images mitochondria. At concentrations above ∼20 μM, the complex images nuclear DNA. Because the complex is extremely photostable, has a large Stokes shift, and displays intrinsic subcellular targeting, its compatibility with super-resolution techniques was investigated. It was found to be very well suited to image mitochondria and nuclear chromatin in two color, 2C-SIM, and STED and 3D-STED, both in fixed and live cells. In particular, due to its vastly improved photostability compared to that of conventional SR probes, it can provide images of nuclear DNA at unprecedented resolution.
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Representative wide field deconvolved images of MCF7 cells stained with MTR (green) and co-stained with [1]Cl4 (red)
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Stomatal opening involves polar, not radial, stiffening of guard cells.
Carter R, Woolfenden H, Baillie A, Amsbury S, Carroll S, Healicon E, Sovatzoglou S, Braybrook S, Gray JE, Hobbs JK, Morris RJ, Fleming AJ.
Current Biology (2017) 27, 2974-2983
Carter R, Woolfenden H, Baillie A, Amsbury S, Carroll S, Healicon E, Sovatzoglou S, Braybrook S, Gray JE, Hobbs JK, Morris RJ, Fleming AJ.
Current Biology (2017) 27, 2974-2983
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Atomic force microscopy force mapping, combined with finite element modelling provide a new insight into the mechanics of stomata in plant leaves. We show that radial thickening of the cell wall is not important for function, over-turning a textbook description, and provide evidence that polar stiffening plays a significant role. Polar stiffening helps to pin the ends of the guard cells when under the action of increased turgor pressure, increasing the extent to which guard cell lengthening leads to pore opening. AFM allows unambiguous identification of the localisation of this stiffening. |
The Synthesis and Photophysical Analysis of a Series of 4-Nitrobenzochalcogenadiazoles for Super-Resolution Microscopy.
Jenkinson DR, Cadby AJ, Jones S.
Chemistry (2017) 23(51):12585-12592.
Jenkinson DR, Cadby AJ, Jones S.
Chemistry (2017) 23(51):12585-12592.
A series of 4-nitrobenzodiazoles with atomic substitution through the chalcogen group were synthesised and their photophysical properties analysed with a view for use in single-molecule localisation microscopy. Sub-diffraction resolution imaging was achieved for silica nanoparticles coated with each dye. Those containing larger atoms were favoured for super-resolution microscopy due to a reduced blink rate (required for stochastic events to be localised). The sulfur-containing molecule was deemed most amenable for widespread use due to the ease of synthetic manipulation compared to the selenium-containing derivative.
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Synthesis of fluorescent nanoparticles. X=O (1), S (2), or Se (3). |
A Novel Application of Non-Destructive Readout Technology to Localisation Microscopy.
Barnett SFH, Snape M, Hunter CN, Juárez MA, Cadby AJ.
Sci Rep. (2017); 7:42313.
Barnett SFH, Snape M, Hunter CN, Juárez MA, Cadby AJ.
Sci Rep. (2017); 7:42313.
The fitting precision in localisation microscopy is highly dependent on the signal to noise ratio. To increase the quality of the image it is therefore important to increase the signal to noise ratio of the measurements. We present an imaging system for localisation microscopy based on non-destructive readout camera technology that can increase the signal to noise ratio of localisation based microscopy. This approach allows for much higher frame rates through subsampling a traditional camera frame. By matching the effective exposure to both the start time and duration of a single molecule we diminish the effects of read noise and temporal noise. We demonstrate the application of this novel method to localisation microscopy and show both an increase in the attainable signal to noise ratio of data collection and an increase in the number of detected events.
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(A) CMOS data, created by subtracting the final NDR frame in a block from the first, equivalent to a 25 fps CMOS image. Data were reconstructed in ThunderSTORM. (B) Events from the NDR data were extracted using custom MATLAB scripts before localisation was performed by fitting to a Gaussian function. Both images are scaled to have the same dynamic range and the scale bar is 1 µm. (C) The SNR values for all the localisations in NDR (blue) and CMOS (red).
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Direct imaging of protein organization in an intact bacterial organelle using high-resolution atomic force microscopy.
Kumar S, Cartron ML, Mullin N, Zian P, Leggett GJ, Hunter CN, Hobbs JK.
ACS Nano (2017) 11, 126-133.
Kumar S, Cartron ML, Mullin N, Zian P, Leggett GJ, Hunter CN, Hobbs JK.
ACS Nano (2017) 11, 126-133.
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This paper shows the detailed protein organisation of an entire bacterial “organelle” – the chromatophore vesicle from Rhodobacter sphaeroides, the entire bacterial photosynthetic system that converts the energy from sunlight into the fuel of life, ATP. It is the first time that such a structure has been imaged in its native state, allowing a detailed statistical analysis of the distribution of light harvesting proteins within the system. The breakthrough in resolution on such a highly curved system (the vesicles are 50 nm spheres) was enabled by use of small amplitude tapping with small AFM cantilevers usually used for high speed imaging, providing improved signal-to-noise and force sensitivity. The figure shows a topographic image of an intact vesicle and the high-pass filtered image clearly revealing the light harvesting protein complexes.
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