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John L. Rubinstein - One of the best experts on this subject based on the ideXlab platform.

  • Through-grid wicking enables high-speed cryoEM Specimen Preparation.
    Acta Crystallographica Section D Structural Biology, 2020
    Co-Authors: Yong Zi Tan, John L. Rubinstein
    Abstract:

    Blotting times for conventional cryoEM Specimen Preparation complicate time-resolved studies and lead to some Specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic Specimen application and through-grid wicking were combined in a high-speed Specimen-Preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a Specimen that suffers from preferred orientations.

  • Through-grid wicking enables high-speed cryoEM Specimen Preparation
    2020
    Co-Authors: Yong Zi Tan, John L. Rubinstein
    Abstract:

    Blotting times for conventional cryoEM Specimen Preparation complicate time-resolved studies and lead to some Specimens adopting preferred orientations or denaturing at the air-water interface. We show that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass fiber filter held against the opposite side, often called the 9back9 of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. We combined ultrasonic Specimen application and through-grid wicking in a high-speed Specimen Preparation device that we name 9Back-it-up9, or BIU. The high liquid-absorption capacity of the glass fiber compared to self-wicking grids appears to make the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice suitable for cryoEM, while the device9s speed reduces adoption of a preferred orientation in a test Specimen.

  • Shake-it-off: a simple ultrasonic cryo-EM Specimen-Preparation device
    Acta Crystallographica Section D Structural Biology, 2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen-Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM Specimen-Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

  • Shake-it-off: A simple ultrasonic cryo-EM Specimen Preparation device
    2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Abstract Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM Specimen Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments. Synopsis A method is presented for high-speed low-volume cryo-EM Specimen Preparation with a device constructed from readily available components.

Timothy Kwok - One of the best experts on this subject based on the ideXlab platform.

  • Shake-it-off: a simple ultrasonic cryo-EM Specimen-Preparation device
    Acta Crystallographica Section D Structural Biology, 2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen-Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM Specimen-Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

  • Shake-it-off: A simple ultrasonic cryo-EM Specimen Preparation device
    2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Abstract Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM Specimen Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments. Synopsis A method is presented for high-speed low-volume cryo-EM Specimen Preparation with a device constructed from readily available components.

Samir Benlekbir - One of the best experts on this subject based on the ideXlab platform.

  • Shake-it-off: a simple ultrasonic cryo-EM Specimen-Preparation device
    Acta Crystallographica Section D Structural Biology, 2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen-Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM Specimen-Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

  • Shake-it-off: A simple ultrasonic cryo-EM Specimen Preparation device
    2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Abstract Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM Specimen Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments. Synopsis A method is presented for high-speed low-volume cryo-EM Specimen Preparation with a device constructed from readily available components.

Hui Guo - One of the best experts on this subject based on the ideXlab platform.

  • Shake-it-off: a simple ultrasonic cryo-EM Specimen-Preparation device
    Acta Crystallographica Section D Structural Biology, 2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen-Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM Specimen-Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

  • Shake-it-off: A simple ultrasonic cryo-EM Specimen Preparation device
    2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Abstract Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM Specimen Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments. Synopsis A method is presented for high-speed low-volume cryo-EM Specimen Preparation with a device constructed from readily available components.

Zev A. Ripstein - One of the best experts on this subject based on the ideXlab platform.

  • Shake-it-off: a simple ultrasonic cryo-EM Specimen-Preparation device
    Acta Crystallographica Section D Structural Biology, 2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen-Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air–water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM Specimen-Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.

  • Shake-it-off: A simple ultrasonic cryo-EM Specimen Preparation device
    2019
    Co-Authors: John L. Rubinstein, Hui Guo, Zev A. Ripstein, Ali Haydaroglu, Christopher M. Yip, Justin M. Di Trani, Samir Benlekbir, Timothy Kwok
    Abstract:

    Abstract Although microscopes and image analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, Specimen Preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here we present a simple cryo-EM Specimen Preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid. The device is controlled by a Raspberry Pi single board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM, rather than instrument design. The simple open-source design permits straightforward customization of the instrument for specialized experiments. Synopsis A method is presented for high-speed low-volume cryo-EM Specimen Preparation with a device constructed from readily available components.