The Experts below are selected from a list of 42780 Experts worldwide ranked by ideXlab platform
Kenji Yasuda - One of the best experts on this subject based on the ideXlab platform.
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origin of individuality of two daughter Cells during the division process examined by the simultaneous measurement of growth and swimming property using an on chip single Cell Cultivation system
Biophysical Journal, 2007Co-Authors: Senkei Umehara, Yuichi Wakamoto, Ippei Inoue, Kenji YasudaAbstract:We examined the origin of individuality of two daughter Cells born from an isolated single Escherichia coli mother Cell during its Cell division process by monitoring the change in its swimming behavior and tumbling frequency using an on-chip single-Cell Cultivation system. By keeping the isolated condition of an observed single Cell, we compared its growth and swimming property within a generation and over up to seven generations. It revealed that running speed decreased as Cell length smoothly increased within each generation, whereas tumbling frequency fluctuated among generations. Also found was an extraordinary tumbling mode characterized by the prolonged duration of pausing in predivisional Cells after Cell constriction. The observed prolonged pausing may imply the coexistence of two distinct control systems in a predivisional Cell, indicating that individuality of daughter Cells emerges after a mother Cell initiates constriction and before it gets physically separated into two new Cell bodies.
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stepwise pattern modification of neuronal network in photo thermally etched agarose architecture on multi electrode array chip for individual Cell based electrophysiological measurement
Lab on a Chip, 2005Co-Authors: Ikurou Suzuki, Yasuhiko Jimbo, Y Sugio, Kenji YasudaAbstract:We have developed a procedure for stepwise topographical control of network patterns and neurite connection directions between adjacent living neurons using an individual-Cell-based on-chip multi-electrode array (MEA) Cell Cultivation system with an agarose microchamber (AMC) array. This procedure enables flexible and precise control of the Cell positions and easy and flexible control of the pattern modification of connections between the Cells in AMCs through stepwise photo-thermal etching in which a portion of the agarose layer on the chip is melted with a 1480 nm infrared laser beam even during Cultivation. With adequate laser power and this stepwise procedue, we can fabricate narrow micrometer-order grooves (microchannels) during Cultivation in a stepwise manner. Using this procedure, we controlled the direction of elongation of axons and dendrites selectively and confirmed the direction by immunostaining. We also demonstrated electrophysiological one-way transmission of signals among aligned hippocampal neurons in which the directions of the neurite connections were controlled using this stepwise photo-thermal etching procedure. These results demonstrate the potential of full direction control of neurite connections between neurons using stepwise photo-thermal etching to form microchannels one by one in an on-chip AMC/MEA Cell Cultivation system. We can thus better understand the meaning of neuronal network patterns and connection directions.
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modification of a neuronal network direction using stepwise photo thermal etching of an agarose architecture
Journal of Nanobiotechnology, 2004Co-Authors: Ikurou Suzuki, Yasuhiko Jimbo, Hiroyuki Moriguchi, Y Sugio, Kenji YasudaAbstract:Control over spatial distribution of individual neurons and the pattern of neural network provides an important tool for studying information processing pathways during neural network formation. Moreover, the knowledge of the direction of synaptic connections between Cells in each neural network can provide detailed information on the relationship between the forward and feedback signaling. We have developed a method for topographical control of the direction of synaptic connections within a living neuronal network using a new type of individual-Cell-based on-chip Cell-Cultivation system with an agarose microchamber array (AMCA). The advantages of this system include the possibility to control positions and number of cultured Cells as well as flexible control of the direction of elongation of axons through stepwise melting of narrow grooves. Such micrometer-order microchannels are obtained by photo-thermal etching of agarose where a portion of the gel is melted with a 1064-nm infrared laser beam. Using this system, we created neural network from individual Rat hippocampal Cells. We were able to control elongation of individual axons during Cultivation (from Cells contained within the AMCA) by non-destructive stepwise photo-thermal etching. We have demonstrated the potential of our on-chip AMCA Cell Cultivation system for the controlled development of individual Cell-based neural networks.
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pattern modification of a neuronal network for individual Cell based electrophysiological measurement using photothermal etching of an agarose architecture with a multielectrode array
IEE proceedings. Nanobiotechnology, 2004Co-Authors: Ikurou Suzuki, Hiroyuki Moriguchi, Kenji Yasuda, Y Sugio, Akihiro Hattori, Yasuhiko JimboAbstract:A new type of individual-Cell-based on-chip multielectrode array (MEA) Cell-Cultivation system with an agarose microchamber (AMC) array for topographical control of the network patterns of a living neuronal network has been developed. The advantages of this system are that it allows control of the Cell positions and numbers for Cultivation using AMCs, as well as easy and flexible control of the pattern of connections between the AMCs through photothermal etching where a portion of the agarose layer is melted with a 1480 nm infrared laser beam. With adequate laser power, narrow micrometer-order grooves (microchannels) can easily be fabricated that can be used to combine neighbouring AMCs to enable topographical control of the neural network pattern. Using this system, an individual-Cell-based neural network pattern was formed of rat hippocampal Cells within the AMC array without Cells escaping from the electrode positions in the microchamber during an eight-day Cultivation, and could record Cell firing in response to 1.5 V, 500 kHz stimulation through an electrode. This demonstrated the potential of the on-chip AMC/MEA Cell Cultivation system for long-term single-Cell-based electrophysiological measurement of a neural network system.
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an agar based on chip neural Cell Cultivation system for stepwise control of network pattern generation during Cultivation
Sensors and Actuators B-chemical, 2004Co-Authors: Yasuhiro Sugio, Hiroyuki Moriguchi, Kazunori Takahashi, Kensuke Kojima, Tomoyuki Kaneko, Kenji YasudaAbstract:We have developed a new type of single-Cell based on-chip Cell-Cultivation system with an agarose microchamber (AMC) array and a photo-thermal etching module for step-by-step topographical control of the network patterns of living neural Cells during long-term Cultivation. The advantages of this system are that (1) it can control positions and numbers of Cells for Cultivation by using agar-based microchambers, and (2) it can change the neural network complexity during Cultivation by photo-thermal melting a portion of agar at the focal point of a 1064 nm infrared laser beam. This laser wavelength is permeable with respect to water and agarose, and it is only absorbed at the thin chromium layer on the chromium-coated glass slide surface at the bottom of the agarose layer. With adequate laser power, we can easily fabricate narrow tunnel-shaped channels between the microchambers at the bottom of the agar layer without the complicated steps conventional microfabrication processes entail even during Cultivation; we demonstrated that rat hippocampal Cells in two adjacent chambers formed fiber connections through new connections between chambers after these had been photo-thermally fabricated. We also verified the fiber connection between those Cells by using calcium-based fluorescent microscopy. These results indicate that this system can potentially be used for studying the complexity of neural network patterns for epigenetic memorization.
Dietrich Kohlheyer - One of the best experts on this subject based on the ideXlab platform.
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analyzing microbial population heterogeneity expanding the toolbox of microfluidic single Cell Cultivations
Journal of Molecular Biology, 2019Co-Authors: Markus Leygeber, Dorina Lindemann, Christian Carsten Sachs, Eugen Kaganovitch, Wolfgang Wiechert, Katharina Nöh, Dietrich KohlheyerAbstract:Abstract Recent research on population heterogeneity revealed fascinating insights into microbial behavior. In particular emerging single-Cell technologies, image-based microfluidics lab-on-chip systems generate insights with spatio-temporal resolution, which are inaccessible with conventional tools. This review reports recent developments and applications of microfluidic single-Cell Cultivation technology, highlighting fields of broad interest such as growth, gene expression and antibiotic resistance and susceptibility. Combining advanced microfluidic single-Cell Cultivation technology for environmental control with automated time-lapse imaging as well as smart computational image analysis offers tremendous potential for novel investigation at the single-Cell level. We propose on-chip control of parameters like temperature, gas supply, pressure or a change in Cultivation mode providing a versatile technology platform to mimic more complex and natural habitats. Digital analysis of the acquired images is a requirement for the extraction of biological knowledge and statistically reliable results demand for robust and automated solutions. Focusing on microbial Cultivations, we compare prominent software systems that emerged during the last decade, discussing their applicability, opportunities and limitations. Next-generation microfluidic devices with a high degree of environmental control combined with time-lapse imaging and automated image analysis will be highly inspiring and beneficial for fruitful interdisciplinary cooperation between microbiologists and microfluidic engineers and image analysts in the field of microbial single-Cell analysis.
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Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions
Frontiers Media S.A., 2018Co-Authors: Joachim Koepff, Christian Carsten Sachs, Wolfgang Wiechert, Katharina Nöh, Dietrich Kohlheyer, Marco Oldiges, Alexander GrünbergerAbstract:Quantitative single-Cell Cultivation has provided fundamental contributions to our understanding of heterogeneity among industrially used microorganisms. Filamentous growing Streptomyces species are emerging platform organisms for industrial production processes, but their exploitation is still limited due to often reported high batch-to-batch variations and unexpected growth and production differences. Population heterogeneity is suspected to be one responsible factor, which is so far not systematically investigated at the single-Cell level. Novel microfluidic single-Cell Cultivation devices offer promising solutions to investigate these phenomena. In this study, we investigated the germination and growth behavior of Streptomyces lividans TK24 under varying medium compositions on different complexity levels (i.e., mycelial growth, hyphal growth and tip elongation) on single-Cell level. Our analysis reveals a remarkable stability within growth and germination of spores and early mycelium development when exposed to constant and defined environments. We show that spores undergo long metabolic adaptation processes of up to > 30 h to adjust to new medium conditions, rather than using a “persister” strategy as a possibility to cope with rapidly changing environments. Due to this uniform behavior, we conclude that S. lividans can be cultivated quite robustly under constant environmental conditions as provided by microfluidic Cultivation approaches. Failure and non-reproducible Cultivations are thus most likely to be found in less controllable larger-scale Cultivation workflows and as a result of environmental gradients within large-scale Cultivations
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RESEARCH ARTICLE Non-Invasive Microbial Metabolic Activity Sensing at Single Cell Level by Perfusion of
2016Co-Authors: Calcein Acetoxymethyl Ester, Katharina Nöh, Abhijeet Singh, Stefan Helfrich, Christina E. M. Krämer, Er Grünberger, Dietrich KohlheyerAbstract:Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a Cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of themetabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensingmethod using a fluorogenic substrate for online monitor-ing of esterase activity and calcein efflux changes in growing wild type bacteria. The fluores-cent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of Cells grown under different conditions at real-time. The dynamic conver-sion of CAM and the active efflux of fluorescent calcein were analyzed by combining microflui-dic single Cell Cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organismCorynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and car-bon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of anti-biotics. Bacteria could be distinguished in growing and non-growing Cells with metabolic activity as well as non-growing and non-fluorescent Cells with no detectable esterase activity. Microfluidic single Cell Cultivation combined with high temporal resolution time-lapse micros-copy facilitated monitoring metabolic activity of stressed Cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAMwith a sampling freemicrofluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria
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non invasive microbial metabolic activity sensing at single Cell level by perfusion of calcein acetoxymethyl ester
PLOS ONE, 2015Co-Authors: Christina Kramer, Wolfgang Wiechert, Katharina Nöh, Alexander Grünberger, Abhijeet Singh, Stefan Helfrich, Dietrich KohlheyerAbstract:Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a Cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of Cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single Cell Cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing Cells with metabolic activity as well as non-growing and non-fluorescent Cells with no detectable esterase activity. Microfluidic single Cell Cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed Cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.
Yukio Nagasaki - One of the best experts on this subject based on the ideXlab platform.
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Design of antioxidative biointerface for separation of hematopoietic stem Cells with high maintenance of undifferentiated phenotype
Journal of Biomedical Materials Research Part A, 2016Co-Authors: Yutaka Ikeda, Tomoki Yoshinari, Hirotoshi Miyoshi, Yukio NagasakiAbstract:During Cell Cultivation, excessively generated reactive oxygen species (ROS) affect Cellular properties and functions. Although Cell Cultivation media contain several types of low-molecular-weight antioxidants, these small antioxidants are internalized into the mitochondria and they disrupt regulated redox balance. Here, we developed a novel biointerface that effectively eliminates ROS on a Cell culture surface. Poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene] (PEG-b-PMNT) was synthesized and covalently coated on a carboxyl group-activated culture dish using sec-amino groups on a PMNT segment followed by immobilization of anti-CD34 antibodies. CD34-positive hematopoietic stem progenitor Cells (HSPCs) were separated from mice fetal liver Cells using our polymer-coated Cell culture dish. The separated HSPCs possessed intact mitochondrial membrane potential compared with those in the conventional Cell Cultivation system. In addition, the expression level of CD34 was maintained for an extended period on our culture dish with the antioxidative biointerface. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2080-2085, 2016.
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a novel biointerface that suppresses Cell morphological changes by scavenging excess reactive oxygen species
Journal of Biomedical Materials Research Part A, 2015Co-Authors: Yutaka Ikeda, Tomoki Yoshinari, Yukio NagasakiAbstract:During Cell Cultivation on conventional culture dishes, various events results in strong stresses that lead to the production of bioactive species such as reactive oxygen species (ROS) and nitric oxide. These reactive species cause variable damage to Cells and stimulate Cellular responses. Here, we report the design of a novel biocompatible surface that decreases stress by not only morphologically modifying the dish surface by using poly(ethylene glycol) tethered chains, but also actively scavenging oxidative stress by using our novel nitroxide radical-containing polymer. A block copolymer, poly(ethylene glycol)-b-poly[(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PEG-b-PMNT) was used to coat the surface of a dish. Differentiation of undifferentiated human leukemia (HL-60) Cells was found to be suppressed on the polymer-coated dish. Notably, HL-60 Cell Cultivation caused apoptosis under high-density conditions, while spontaneous apoptosis was suppressed in Cells plated on the PEG-b-PMNT-modified surface, because a healthy mitochondrial membrane potential was maintained. In contrast, low molecular weight antioxidants did not have apparent effects on the maintenance of mitochondria. We attribute this to the lack of Cellular internalization of our immobilized polymer and selective scavenging of excessive ROS generated outside of Cells. These results demonstrate the utility of our novel biocompatible material for actively scavenging ROS and thus maintaining Cellular morphology.
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a novel biointerface that suppresses Cell morphological changes by scavenging excess reactive oxygen species
Journal of Biomedical Materials Research Part A, 2015Co-Authors: Yutaka Ikeda, Tomoki Yoshinari, Yukio NagasakiAbstract:During Cell Cultivation on conventional culture dishes, various events results in strong stresses that lead to the production of bioactive species such as reactive oxygen species (ROS) and nitric oxide. These reactive species cause variable damage to Cells and stimulate Cellular responses. Here, we report the design of a novel biocompatible surface that decreases stress by not only morphologically modifying the dish surface by using poly(ethylene glycol) tethered chains, but also actively scavenging oxidative stress by using our novel nitroxide radical-containing polymer. A block copolymer, poly(ethylene glycol)-b-poly[(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PEG-b-PMNT) was used to coat the surface of a dish. Differentiation of undifferentiated human leukemia (HL-60) Cells was found to be suppressed on the polymer-coated dish. Notably, HL-60 Cell Cultivation caused apoptosis under high-density conditions, while spontaneous apoptosis was suppressed in Cells plated on the PEG-b-PMNT-modified surface, because a healthy mitochondrial membrane potential was maintained. In contrast, low molecular weight antioxidants did not have apparent effects on the maintenance of mitochondria. We attribute this to the lack of Cellular internalization of our immobilized polymer and selective scavenging of excessive ROS generated outside of Cells. These results demonstrate the utility of our novel biocompatible material for actively scavenging ROS and thus maintaining Cellular morphology. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2815–2822, 2015.
Alexander Grünberger - One of the best experts on this subject based on the ideXlab platform.
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dMSCC: a microfluidic platform for microbial single-Cell Cultivation of Corynebacterium glutamicum under dynamic environmental medium conditions
Lab on a chip, 2020Co-Authors: Sarah Täuber, Corinna Golze, Eric Von Lieres, Alexander GrünbergerAbstract:In nature and in technical systems, microbial Cells are often exposed to rapidly fluctuating environmental conditions. These conditions can vary in quality, e.g., the existence of a starvation zone, and quantity, e.g., the average residence time in this zone. For strain development and process design, Cellular response to such fluctuations needs to be systematically analysed. However, the existing methods for physically imitating rapidly changing environmental conditions are limited in spatio-temporal resolution. Hence, we present a novel microfluidic system for Cultivation of single Cells and small Cell clusters under dynamic environmental conditions (dynamic microfluidic single-Cell Cultivation (dMSCC)). This system enables the control of nutrient availability and composition between two media with second to minute resolution. We validate our technology using the industrially relevant model organism Corynebacterium glutamicum. The organism was exposed to different oscillation frequencies between nutrient excess (feasts) and scarcity (famine). The resulting changes in Cellular physiology, such as the colony growth rate and Cell morphology, were analysed and revealed significant differences in the growth rate and Cell length between the different conditions. dMSCC also allows the application of defined but randomly changing nutrient conditions, which is important for reproducing more complex conditions from natural habitats and large-scale bioreactors. The presented system lays the foundation for the Cultivation of Cells under complex changing environmental conditions.
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Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions
Frontiers Media S.A., 2018Co-Authors: Joachim Koepff, Christian Carsten Sachs, Wolfgang Wiechert, Katharina Nöh, Dietrich Kohlheyer, Marco Oldiges, Alexander GrünbergerAbstract:Quantitative single-Cell Cultivation has provided fundamental contributions to our understanding of heterogeneity among industrially used microorganisms. Filamentous growing Streptomyces species are emerging platform organisms for industrial production processes, but their exploitation is still limited due to often reported high batch-to-batch variations and unexpected growth and production differences. Population heterogeneity is suspected to be one responsible factor, which is so far not systematically investigated at the single-Cell level. Novel microfluidic single-Cell Cultivation devices offer promising solutions to investigate these phenomena. In this study, we investigated the germination and growth behavior of Streptomyces lividans TK24 under varying medium compositions on different complexity levels (i.e., mycelial growth, hyphal growth and tip elongation) on single-Cell level. Our analysis reveals a remarkable stability within growth and germination of spores and early mycelium development when exposed to constant and defined environments. We show that spores undergo long metabolic adaptation processes of up to > 30 h to adjust to new medium conditions, rather than using a “persister” strategy as a possibility to cope with rapidly changing environments. Due to this uniform behavior, we conclude that S. lividans can be cultivated quite robustly under constant environmental conditions as provided by microfluidic Cultivation approaches. Failure and non-reproducible Cultivations are thus most likely to be found in less controllable larger-scale Cultivation workflows and as a result of environmental gradients within large-scale Cultivations
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non invasive microbial metabolic activity sensing at single Cell level by perfusion of calcein acetoxymethyl ester
PLOS ONE, 2015Co-Authors: Christina Kramer, Wolfgang Wiechert, Katharina Nöh, Alexander Grünberger, Abhijeet Singh, Stefan Helfrich, Dietrich KohlheyerAbstract:Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a Cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of Cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single Cell Cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing Cells with metabolic activity as well as non-growing and non-fluorescent Cells with no detectable esterase activity. Microfluidic single Cell Cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed Cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.
Wolfgang Wiechert - One of the best experts on this subject based on the ideXlab platform.
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analyzing microbial population heterogeneity expanding the toolbox of microfluidic single Cell Cultivations
Journal of Molecular Biology, 2019Co-Authors: Markus Leygeber, Dorina Lindemann, Christian Carsten Sachs, Eugen Kaganovitch, Wolfgang Wiechert, Katharina Nöh, Dietrich KohlheyerAbstract:Abstract Recent research on population heterogeneity revealed fascinating insights into microbial behavior. In particular emerging single-Cell technologies, image-based microfluidics lab-on-chip systems generate insights with spatio-temporal resolution, which are inaccessible with conventional tools. This review reports recent developments and applications of microfluidic single-Cell Cultivation technology, highlighting fields of broad interest such as growth, gene expression and antibiotic resistance and susceptibility. Combining advanced microfluidic single-Cell Cultivation technology for environmental control with automated time-lapse imaging as well as smart computational image analysis offers tremendous potential for novel investigation at the single-Cell level. We propose on-chip control of parameters like temperature, gas supply, pressure or a change in Cultivation mode providing a versatile technology platform to mimic more complex and natural habitats. Digital analysis of the acquired images is a requirement for the extraction of biological knowledge and statistically reliable results demand for robust and automated solutions. Focusing on microbial Cultivations, we compare prominent software systems that emerged during the last decade, discussing their applicability, opportunities and limitations. Next-generation microfluidic devices with a high degree of environmental control combined with time-lapse imaging and automated image analysis will be highly inspiring and beneficial for fruitful interdisciplinary cooperation between microbiologists and microfluidic engineers and image analysts in the field of microbial single-Cell analysis.
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Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions
Frontiers Media S.A., 2018Co-Authors: Joachim Koepff, Christian Carsten Sachs, Wolfgang Wiechert, Katharina Nöh, Dietrich Kohlheyer, Marco Oldiges, Alexander GrünbergerAbstract:Quantitative single-Cell Cultivation has provided fundamental contributions to our understanding of heterogeneity among industrially used microorganisms. Filamentous growing Streptomyces species are emerging platform organisms for industrial production processes, but their exploitation is still limited due to often reported high batch-to-batch variations and unexpected growth and production differences. Population heterogeneity is suspected to be one responsible factor, which is so far not systematically investigated at the single-Cell level. Novel microfluidic single-Cell Cultivation devices offer promising solutions to investigate these phenomena. In this study, we investigated the germination and growth behavior of Streptomyces lividans TK24 under varying medium compositions on different complexity levels (i.e., mycelial growth, hyphal growth and tip elongation) on single-Cell level. Our analysis reveals a remarkable stability within growth and germination of spores and early mycelium development when exposed to constant and defined environments. We show that spores undergo long metabolic adaptation processes of up to > 30 h to adjust to new medium conditions, rather than using a “persister” strategy as a possibility to cope with rapidly changing environments. Due to this uniform behavior, we conclude that S. lividans can be cultivated quite robustly under constant environmental conditions as provided by microfluidic Cultivation approaches. Failure and non-reproducible Cultivations are thus most likely to be found in less controllable larger-scale Cultivation workflows and as a result of environmental gradients within large-scale Cultivations
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non invasive microbial metabolic activity sensing at single Cell level by perfusion of calcein acetoxymethyl ester
PLOS ONE, 2015Co-Authors: Christina Kramer, Wolfgang Wiechert, Katharina Nöh, Alexander Grünberger, Abhijeet Singh, Stefan Helfrich, Dietrich KohlheyerAbstract:Phase contrast microscopy cannot give sufficient information on bacterial metabolic activity, or if a Cell is dead, it has the fate to die or it is in a viable but non-growing state. Thus, a reliable sensing of the metabolic activity helps to distinguish different categories of viability. We present a non-invasive instantaneous sensing method using a fluorogenic substrate for online monitoring of esterase activity and calcein efflux changes in growing wild type bacteria. The fluorescent conversion product of calcein acetoxymethyl ester (CAM) and its efflux indicates the metabolic activity of Cells grown under different conditions at real-time. The dynamic conversion of CAM and the active efflux of fluorescent calcein were analyzed by combining microfluidic single Cell Cultivation technology and fluorescence time lapse microscopy. Thus, an instantaneous and non-invasive sensing method for apparent esterase activity was created without the requirement of genetic modification or harmful procedures. The metabolic activity sensing method consisting of esterase activity and calcein secretion was demonstrated in two applications. Firstly, growing colonies of our model organism Corynebacterium glutamicum were confronted with intermittent nutrient starvation by interrupting the supply of iron and carbon, respectively. Secondly, bacteria were exposed for one hour to fatal concentrations of antibiotics. Bacteria could be distinguished in growing and non-growing Cells with metabolic activity as well as non-growing and non-fluorescent Cells with no detectable esterase activity. Microfluidic single Cell Cultivation combined with high temporal resolution time-lapse microscopy facilitated monitoring metabolic activity of stressed Cells and analyzing their descendants in the subsequent recovery phase. Results clearly show that the combination of CAM with a sampling free microfluidic approach is a powerful tool to gain insights in the metabolic activity of growing and non-growing bacteria.