Connecting μ-fluidics to electron microscopy

Kemmerling, Simon; Ziegler, Jörg C.; Schweighauser, Gabriel; Arnold, Stefan; Giss, Dominic; Müller, Shirley A.; Ringler, Philippe; Goldie, Kenneth N.; Goedecke, Nils; Hierlemann, Andreas; Stahlberg, Henning; Engel, Andréas; Braun, Thomas

doi:10.1016/j.jsb.2011.11.001

Cited by 25 publications

(43 citation statements)

References 23 publications

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“…A typical scenario describing how openBEB is used to combine live cell microscopy, microfluidic control and a new approach for visual proteomics called “spread and lyse” [8,9], is then presented.…”

Section: Resultsmentioning

confidence: 99%

“…The use of openBEB together with our recent hardware developments that connect micro-fluidics to EM for visual proteomics [8,10] is reported. In the demonstration experiment adherent eukaryotic cells are cultured, and monitored by time-lapse LM throughout.…”

Section: Resultsmentioning

confidence: 99%

“…At specific time points, pulse chase investigations are performed and, after a chase time, individual cells are lysed by electroporation [10]. Subsequently, the cell lysate is transferred into a microcapillary, prepared for EM and imaged for analysis by “lyse and spread visual proteomics” [8,9]. The presentation focuses on the application of openBEB using the GUI, and demonstrates some typical aspects of the program.…”

Section: Resultsmentioning

confidence: 99%

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openBEB: open biological experiment browser for correlative measurements

et al. 2014

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BackgroundNew experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis.ResultsWe have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository.ConclusionsThe versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the “house keeping” abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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openBEB: open biological experiment browser for correlative measurements

et al. 2014

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“…In this context, visual proteomics aims at identifying the proteins and interaction networks in the recorded tomogram . Since ET is limited to very thin (1 μm or below) cells or sections of cells obtained, e.g., by lamella milling, and the process of data acquisition and analysis is very time consuming and cumbersome, a complementary lyse‐and‐spread visual proteomics approach was developed . This method uses a single‐cell lysis device combined with microcapillary sample deposition to collect the lysate of a single cell, condition it, and spread it over a grid for imaging by negative stain EM .…”

Section: New Opportunities and Outlookmentioning

confidence: 99%

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”

et al. 2018

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This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

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“…It significantly reduces sample consumption and improves user-control over sample preparation as a whole. Furthermore, the method allows novel experimental applications; such as the preparation of isolated biological components of individual cells in an approach called "single cell visual proteomics" 22 23 24 25 .…”

Section: Introductionmentioning

confidence: 99%

Miniaturized Sample Preparation for Transmission Electron Microscopy

Schmidli

Rima

Arnold

et al. 2018

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Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for "visual proteomics," or "lossless" total sample preparation for quantitative analysis of complex samples.

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Connecting μ-fluidics to electron microscopy

Cited by 25 publications

References 23 publications

openBEB: open biological experiment browser for correlative measurements

openBEB: open biological experiment browser for correlative measurements

Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”

Miniaturized Sample Preparation for Transmission Electron Microscopy

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