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We develop here a novel modelling approach with the aim of closing the conceptual gap between tumour-level metabolic processes and the metabolic processes occurring in individual cancer cells. In particular, the metabolism in hepatocellular carcinoma derived cell lines (HEPG2 cells) has been well characterized but implementations of multiscale models integrating this known metabolism have not been previously reported. We therefore extend a previously published multiscale model of vascular tumour growth, and integrate it with an experimentally verified network of central metabolism in HEPG2 cells. This resultant combined model links spatially heterogeneous vascular tumour growth with known metabolic networks within tumour cells and accounts for blood flow, angiogenesis, vascular remodelling and nutrient/growth factor transport within a growing tumour, as well as the movement of, and interactions between normal and cancer cells. Model simulations report for the first time, predictions of spatially resolved time courses of core metabolites in HEPG2 cells. These simulations can be performed at a sufficient scale to incorporate clinically relevant features of different tumour systems using reasonable computational resources. Our results predict larger than expected temporal and spatial heterogeneity in the intracellular concentrations of glucose, oxygen, lactate pyruvate, f16bp and Acetyl-CoA. The integrated multiscale model developed here provides an ideal quantitative framework in which to study the relationship between dosage, timing, and scheduling of anti-neoplastic agents and the physiological effects of tumour metabolism at the cellular level. Such models, therefore, have the potential to inform treatment decisions when drug response is dependent on the metabolic state of individual cancer cells.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Neoplasias , Neoplasias Vasculares , Simulación por Computador , Humanos , Modelos Biológicos , Neoplasias/patologíaRESUMEN
A dedicated stimulated emission depletion (STED) microscope had been designed and implemented into the Göttingen Instrument for Nano-Imaging with X-rays (GINIX) at the synchrotron beamline P10 of the PETRAâ III storage ring (DESY, Hamburg). The microscope was installed on the same optical table used for X-ray holography and scanning small-angle X-ray scattering (SAXS). Scanning SAXS was implemented with the Kirkpatrick-Baez (KB) nano-focusing optics of GINIX, while X-ray holography used a combined KB and X-ray waveguide optical system for full-field projection recordings at a defocus position of the object. The STED optical axis was aligned (anti-)parallel to the focused synchrotron beam and was laterally displaced from the KB focus. This close proximity between the STED and the X-ray probe enabled in situ combined recordings on the same biological cell, tissue or any other biomolecular sample, using the same environment and mounting. Here, the instrumentation and experimental details of this correlative microscopy approach are described, as first published in our preceding work [Bernhardt et al. (2018), Nat. Commun. 9, 3641], and the capabilities of correlative STED microscopy, X-ray holography and scanning SAXS are illustrated by presenting additional datasets on cardiac tissue cells with labeled actin cytoskeleton.
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Microscopía/instrumentación , Rayos X , Prueba de Estudio Conceptual , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.
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Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Microtúbulos/metabolismo , EstereoisomerismoRESUMEN
Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 µm.
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Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Agua , Artefactos , Células Cultivadas , Fibroblastos/citología , Colorantes Fluorescentes , Compuestos de Oro , Humanos , Nanopartículas del Metal , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Aceites , Poliestirenos , RefractometríaRESUMEN
Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113-7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (µmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 µm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 µm. The size of the bud linearly depends on µmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on µmax. There are two temperature regions (5-31°C vs. 33-40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33-40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the µmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.
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Tamaño de la Célula , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Temperatura , Anaerobiosis , Biomasa , Reactores Biológicos , Ciclo Celular , División Celular , Citometría de Flujo , Glucosa/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
The concepts called STED/RESOLFT superresolve features by a light-driven transfer of closely packed molecules between two different states, typically a nonfluorescent "off" state and a fluorescent "on" state at well-defined coordinates on subdiffraction scales. For this, the applied light intensity must be sufficient to guarantee the state difference for molecules spaced at the resolution sought. Relatively high intensities have therefore been applied throughout the imaging to obtain the highest resolutions. At regions where features are far enough apart that molecules could be separated with lower intensity, the excess intensity just adds to photobleaching. Here, we introduce DyMIN (standing for Dynamic Intensity Minimum) scanning, generalizing and expanding on earlier concepts of RESCue and MINFIELD to reduce sample exposure. The principle of DyMIN is that it only uses as much on/off-switching light as needed to image at the desired resolution. Fluorescence can be recorded at those positions where fluorophores are found within a subresolution neighborhood. By tuning the intensity (and thus resolution) during the acquisition of each pixel/voxel, we match the size of this neighborhood to the structures being imaged. DyMIN is shown to lower the dose of STED light on the scanned region up to â¼20-fold under common biological imaging conditions, and >100-fold for sparser 2D and 3D samples. The bleaching reduction can be converted into accordingly brighter images at <30-nm resolution.
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A powerful approach for the optimization of industrial bioprocesses is to perform detailed simulations integrating large-scale computational fluid dynamics (CFD) and cellular reaction dynamics (CRD). However, complex metabolic kinetic models containing a large number of equations pose formidable challenges in CFD-CRD coupling and computation time afterward. This necessitates to formulate a relatively simple but yet representative model structure. Such a kinetic model should be able to reproduce metabolic responses for short-term (mixing time scale of tens of seconds) and long-term (fed-batch cultivation of hours/days) dynamics in industrial bioprocesses. In this paper, we used Penicillium chrysogenum as a model system and developed a metabolically structured kinetic model for growth and production. By lumping the most important intracellular metabolites in 5 pools and 4 intracellular enzyme pools, linked by 10 reactions, we succeeded in maintaining the model structure relatively simple, while providing informative insight into the state of the organism. The performance of this 9-pool model was validated with a periodic glucose feast-famine cycle experiment at the minute time scale. Comparison of this model and a reported black box model for this strain shows the necessity of employing a structured model under feast-famine conditions. This proposed model provides deeper insight into the in vivo kinetics and, most importantly, can be straightforwardly integrated into a computational fluid dynamic framework for simulating complete fermentation performance and cell population dynamics in large scale and small scale fermentors. Biotechnol. Bioeng. 2017;114: 1733-1743. © 2017 Wiley Periodicals, Inc.
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Proliferación Celular/fisiología , Glucosa/metabolismo , Análisis de Flujos Metabólicos/métodos , Redes y Vías Metabólicas/fisiología , Modelos Biológicos , Penicillium chrysogenum/fisiología , Simulación por Computador , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Cinética , Tasa de Depuración Metabólica/fisiología , Complejos Multienzimáticos/metabolismo , Penicillium chrysogenum/citología , Factores de TiempoRESUMEN
We develop an off-lattice, agent-based model to describe vasculogenesis, the de novo formation of blood vessels from endothelial progenitor cells during development. The endothelial cells that comprise our vessel network are viewed as linearly elastic spheres that move in response to the forces they experience. We distinguish two types of endothelial cells: vessel elements are contained within the network and tip cells are located at the ends of vessels. Tip cells move in response to mechanical forces caused by interactions with neighbouring vessel elements and the local tissue environment, chemotactic forces and a persistence force which accounts for their tendency to continue moving in the same direction. Vessel elements are subject to similar mechanical forces but are insensitive to chemotaxis. An angular persistence force representing interactions with the local tissue is introduced to stabilise buckling instabilities caused by cell proliferation. Only vessel elements proliferate, at rates which depend on their degree of stretch: elongated elements have increased rates of proliferation, and compressed elements have reduced rates. Following division, the fate of the new cell depends on the local mechanical environment: the probability of forming a new sprout is increased if the parent vessel is highly compressed and the probability of being incorporated into the parent vessel increased if the parent is stretched. Simulation results reveal that our hybrid model can reproduce the key qualitative features of vasculogenesis. Extensive parameter sensitivity analyses show that significant changes in network size and morphology are induced by varying the chemotactic sensitivity of tip cells, and the sensitivities of the proliferation rate and the sprouting probability to mechanical stretch. Varying the chemotactic sensitivity directly influences the directionality of the networks. The degree of branching, and thereby the density of the networks, is influenced by the sprouting probability. Glyphs that simultaneously depict several network properties are introduced to show how these and other network quantities change over time and also as model parameters vary. We also show how equivalent glyphs constructed from in vivo data could be used to discriminate between normal and tumour vasculature and, in the longer term, for model validation. We conclude that our biomechanical hybrid model can generate vascular networks that are qualitatively similar to those generated from in vitro and in vivo experiments.
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División Celular , Quimiotaxis , Células Endoteliales , Modelos Cardiovasculares , Neoplasias , Neovascularización Patológica , Animales , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Neoplasias/química , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , RatasRESUMEN
The trajectories, referred to as lifelines, of individual microorganisms in an industrial scale fermentor under substrate limiting conditions were studied using an Euler-Lagrange computational fluid dynamics approach. The metabolic response to substrate concentration variations along these lifelines provides deep insight in the dynamic environment inside a large-scale fermentor, from the point of view of the microorganisms themselves. We present a novel methodology to evaluate this metabolic response, based on transitions between metabolic "regimes" that can provide a comprehensive statistical insight in the environmental fluctuations experienced by microorganisms inside an industrial bioreactor. These statistics provide the groundwork for the design of representative scale-down simulators, mimicking substrate variations experimentally. To focus on the methodology we use an industrial fermentation of Penicillium chrysogenum in a simplified representation, dealing with only glucose gradients, single-phase hydrodynamics, and assuming no limitation in oxygen supply, but reasonably capturing the relevant timescales. Nevertheless, the methodology provides useful insight in the relation between flow and component fluctuation timescales that are expected to hold in physically more thorough simulations. Microorganisms experience substrate fluctuations at timescales of seconds, in the order of magnitude of the global circulation time. Such rapid fluctuations should be replicated in truly industrially representative scale-down simulators.
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Soil microorganisms mineralize lignin-derived aromatic carbon sources using oxidative catabolic pathways, such as the ß-ketoadipate pathway. Although this aromatic pathway is one of the best-studied pathways in biochemistry, the complete pathway, including its regulation by aromatic carbon sources, has not been integrated into the metabolic network. In particular, information about the in vivo operation (e.g., kinetics and flux capacity) of the pathway is lacking. In this contribution, we use kinetic modeling and thermodynamic analysis to evaluate the in vivo operation of this key aromatic multi-step pathway. The resulting ab initio deterministic model of benzoate degradation via the ß-ketoadipate (ortho-cleavage) pathway in Pseudomonas putida KT2440 is presented. The kinetic model includes mechanistic rate expressions for the enzymes and transport processes. The design and experimental validation of the model are driven by data generated from short-term perturbation experiments in a benzoate-limited continuous culture. The results of rigorous modeling of the in vivo dynamics provide strong support for flux regulation by the benzoate transporter and the enzymes forming and cleaving catechol. Revisiting the ß-ketoadipate pathway might be valuable for applications in different fields, such as biochemistry and metabolic engineering, that use lignin monomers as a carbon source.
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Canonized view on temperature effects on growth rate of microorganisms is based on assumption of protein denaturation, which is not confirmed experimentally so far. We develop an alternative concept, which is based on view that limits of thermal tolerance are based on imbalance of cellular energy allocation. Therefore, we investigated growth suppression of yeast Saccharomyces cerevisiae in the supraoptimal temperature range (30-40°C), i.e. above optimal temperature (Topt). The maximal specific growth rate (µmax) of biomass, its concentration and yield on glucose (Yx/glc) were measured across the whole thermal window (5-40°C) of the yeast in batch anaerobic growth on glucose. Specific rate of glucose consumption, specific rate of glucose consumption for maintenance (mglc), true biomass yield on glucose (Yx/glc(true)), fractional conservation of substrate carbon in product and ATP yield on glucose (Yatp/glc) were estimated from the experimental data. There was a negative linear relationship between ATP, ADP and AMP concentrations and specific growth rate at any growth conditions, whilst the energy charge was always high (~0.83). There were two temperature regions where mglc differed 12-fold, which points to the existence of a 'low' (within 5-31°C) and a 'high' (within 33-40°C) metabolic mode regarding maintenance requirements. The rise from the low to high mode occurred at 31-32°C in step-wise manner and it was accompanied with onset of suppression of µmax. High mglc at supraoptimal temperatures indicates a significant reduction of scope for growth, due to high maintenance cost. Analysis of temperature dependencies of product formation efficiency and Yatp/glc revealed that the efficiency of energy metabolism approaches its lower limit at 26-31°C. This limit is reflected in the predetermined combination of Yx/glc(true), elemental biomass composition and degree of reduction of the growth substrate. Approaching the limit implies a reduction of the safety margin of metabolic efficiency. We hypothesize that a temperature increase above Topt (e.g. >31°C) triggers both an increment in mglc and suppression of µmax, which together contribute to an upshift of Yatp/glc from the lower limit and thus compensate for the loss of the safety margin. This trade-off allows adding 10 more degrees to Topt and extends the thermal window up to 40°C, sustaining survival and reproduction in supraoptimal temperatures. Deeper understanding of the limits of thermal tolerance can be practically exploited in biotechnological applications.
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Biomasa , Saccharomyces cerevisiae/metabolismo , Temperatura , Nucleótidos de Adenina/metabolismo , Adenosina Trifosfato/metabolismo , Algoritmos , Anaerobiosis , Costos y Análisis de Costo , Metabolismo Energético , Etanol/análisis , Etanol/metabolismo , Glucosa/metabolismo , Cinética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.
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Hepatocitos/enzimología , Hepatocitos/metabolismo , Espectrometría de Masas/métodos , Péptidos/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Anticuerpos/inmunología , Afinidad de Anticuerpos , Hidrocarburo de Aril Hidroxilasas/inmunología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Atorvastatina/farmacocinética , Células Cultivadas , Cromatografía Liquida/métodos , Citocromo P-450 CYP3A/inmunología , Citocromo P-450 CYP3A/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Hepatocitos/citología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Péptidos/inmunología , Pravastatina/farmacocinética , Cultivo Primario de Células , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Norbin is a neuron-specific, cytosolic protein that interacts with the metabotropic glutamate receptor 5 (mGluR5) and has a profound impact on mGluR5 signaling. Yet, little is known about its synaptic distribution. RESULTS: Here we have analyzed the spatial relationship between Norbin, postsynaptic density protein 95 (PSD-95), actin and mGluR5 in spines using super-resolution microscopy. Norbin was found to have a high degree of colocalization with actin and a lower degree of colocalization with PSD-95. Co-immunoprecipitation studies confirmed that interaction occurs between Norbin and actin, but not between Norbin and PSD-95. Norbin was also found to have a high degree of colocalization with the perisynaptically located mGluR5. Findings based on structured illumination microscopy (3D-SIM) of exogenous expressed Norbin-GFP were confirmed by stimulated emission depletion microscopy (STED) of immunolabeled endogenous Norbin. CONCLUSIONS: Norbin associates with actin rather than with PSD-95 in dendritic spines. Results regarding protein localization and colocalization performed with conventional confocal microscopy must be interpreted with great caution. The now available super-resolution microscopy techniques provide more accurate information about sub-cellular protein localization than previously was possible.
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Actinas/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Neuropéptidos/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Células Cultivadas , Femenino , Masculino , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP.
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Adenina/metabolismo , Adenosina Trifosfato/metabolismo , Metabolismo Energético , Glucosa/metabolismo , Saccharomyces cerevisiae/metabolismo , Biología de Sistemas , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Fructosadifosfatos/metabolismo , Glucólisis , Hidrólisis , Modelos Biológicos , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de TiempoRESUMEN
Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.
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Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Núcleo Celular/virología , Dependovirus/metabolismo , Infecciones por Parvoviridae/virología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Línea Celular , Núcleo Celular/metabolismo , Dependovirus/química , Dependovirus/genética , Humanos , Datos de Secuencia Molecular , Infecciones por Parvoviridae/metabolismo , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
The membrane trafficking machinery provides a transport and sorting system for many cellular proteins. We propose a mechanistic agent-based computer simulation to integrate and test the hypothesis of vesicle transport embedded into a detailed model cell. The method tracks both the number and location of the vesicles. Thus both the stochastic properties due to the low numbers and the spatial aspects are preserved. The underlying molecular interactions that control the vesicle actions are included in a multi-scale manner based on the model of Heinrich and Rapoport (2005). By adding motor proteins we can improve the recycling process of SNAREs and model cell polarization. Our model also predicts that coat molecules should have a high turnover at the compartment membranes, while the turnover of motor proteins has to be slow. The modular structure of the underlying model keeps it tractable despite the overall complexity of the vesicle system. We apply our model to receptor-mediated endocytosis and show how a polarized cytoskeleton structure leads to polarized distributions in the plasma membrane both of SNAREs and the Ste2p receptor in yeast. In addition, we can couple signal transduction and membrane trafficking steps in one simulation, which enables analyzing the effect of receptor-mediated endocytosis on signaling.
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Citoesqueleto/metabolismo , Modelos Biológicos , Membrana Celular/metabolismo , Polaridad Celular , Simulación por Computador , Endocitosis , Exocitosis , Cinética , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , Transducción de SeñalRESUMEN
The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25-250 times faster recordings.DOI:http://dx.doi.org/10.7554/eLife.00248.001.
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Células Epiteliales/ultraestructura , Proteínas Fluorescentes Verdes/genética , Microscopía Fluorescente/métodos , Animales , Línea Celular , Células Epiteliales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Riñón/metabolismo , Riñón/ultraestructura , Luz , Macropodidae , Microscopía Fluorescente/instrumentación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
In the field of metabolomics, GC-MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC-MS techniques. Only few groups have so far adopted GC-MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography-isotope dilution mass spectrometry (GC-IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available (13)C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.
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Cromatografía de Gases y Espectrometría de Masas/métodos , Marcaje Isotópico/métodos , Metabolómica/métodos , Anaerobiosis , Isótopos de Carbono/análisis , Extractos Celulares/química , Chlorophyta , Diseño de Equipo , Espacio Extracelular/química , Espacio Intracelular/química , Metabolómica/normas , Oximas/metabolismo , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Compuestos de TrimetilsililoRESUMEN
We introduce a parallelized STED microscope featuring m = 4 pairs of scanning excitation and STED beams, providing m-fold increased imaging speed of a given sample area, while maintaining basically all of the advantages of single beam scanning. Requiring only a single laser source and fiber input, the setup is inherently aligned both spatially and temporally. Given enough laser power, the design is readily scalable to higher degrees of parallelization m.
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Tecnología de Fibra Óptica/instrumentación , Aumento de la Imagen/instrumentación , Microscopía Confocal/instrumentación , Microscopía Fluorescente/instrumentación , Nanotecnología/instrumentación , Refractometría/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.