scholarly journals Shake-it-off: a simple ultrasonic cryo-EM specimen-preparation device

2019 ◽  
Vol 75 (12) ◽  
pp. 1063-1070 ◽  
Author(s):  
John L. Rubinstein ◽  
Hui Guo ◽  
Zev A. Ripstein ◽  
Ali Haydaroglu ◽  
Aaron Au ◽  
...  
Keyword(s):  
Thin Film ◽  
Raspberry Pi ◽  
Specimen Preparation ◽  
Analysis Software ◽  
Preparation Methods ◽  
Time Resolved ◽  
Transfer Protein ◽  
Air Water Interface ◽  
Sample Application ◽  
Ultrasonic Humidifier

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.

scholarly journals Shake-it-off: A simple ultrasonic cryo-EM specimen preparation device

2019 ◽  
Author(s):  
John L. Rubinstein ◽  
Hui Guo ◽  
Zev A. Ripstein ◽  
Ali Haydaroglu ◽  
Aaron Au ◽  
...  
Keyword(s):  
Thin Film ◽  
High Speed ◽  
Raspberry Pi ◽  
Specimen Preparation ◽  
Preparation Methods ◽  
Time Resolved ◽  
Transfer Protein ◽  
Air Water Interface ◽  
Sample Application ◽  
Ultrasonic Humidifier

AbstractAlthough 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.SynopsisA method is presented for high-speed low-volume cryo-EM specimen preparation with a device constructed from readily available components.

scholarly journals Through-grid wicking enables high-speed cryoEM specimen preparation

2020 ◽  
Vol 76 (11) ◽  
pp. 1092-1103
Author(s):  
Yong Zi Tan ◽  
John L. Rubinstein
Keyword(s):  
Glass Fiber ◽  
High Speed ◽  
Protein Complexes ◽  
Absorption Capacity ◽  
Specimen Preparation ◽  
Water Interface ◽  
Time Resolved ◽  
Glass Fiber Filter ◽  
Liquid Absorption ◽  
Air Water Interface

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.

scholarly journals Particle Lengths of Whitefly-Transmitted Criniviruses

Plant Disease ◽  
2000 ◽  
Vol 84 (7) ◽  
pp. 803-805 ◽  
Author(s):  
H.-Y. Liu ◽  
G. C. Wisler ◽  
J. E. Duffus
Keyword(s):  
Specimen Preparation ◽  
Analysis Software ◽  
Preparation Methods ◽  
Particle Measurements ◽  
Lettuce Infectious Yellows Virus ◽  
Capture Method ◽  
Antibody Capture ◽  
Tomato Chlorosis Virus ◽  
Tomato Infectious Chlorosis Virus ◽  
Particle Length

An improved method for particle length measurement was used for six members of the genus Crinivirus. Particle measurements were conducted with a CCD-72S solid state camera, which was interfaced with a Zeiss EM 109 electron microscope, and analyzed using the analysis 2.1 Image Analysis Software. In comparisons of specimen preparation methods, the leaf dip method is more representative and reproducible than the antibody capture method or preparation from purified virions. Particle length (nm) ranges of whitefly-transmitted criniviruses are: Abutilon yellows virus (AYV), 800 to 850; Cucurbit yellow stunting disorder virus (CYSDV), 750 to 800; Lettuce chlorosis virus (LCV), 800 to 850; Lettuce infectious yellows virus (LIYV), 700 to 750; Tomato chlorosis virus (ToCV), 800 to 850; and Tomato infectious chlorosis virus (TICV), 850 to 900.

scholarly journals Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

2020 ◽  
Author(s):  
David P. Klebl ◽  
Molly S. C. Gravett ◽  
Dimitrios Kontziampasis ◽  
David J. Wright ◽  
Robin S. Bon ◽  
...  
Keyword(s):  
Sample Preparation ◽  
New Technologies ◽  
Water Interface ◽  
Time Resolved ◽  
Macromolecular Complexes ◽  
Air Water Interface ◽  
Sample Application ◽  
Silver Bullet ◽  
Particle Behaviour

AbstractA host of new technologies are under development to improve the quality and reproducibility of cryoEM grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot™, chameleon and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms, (the fastest reported to date), to interrogate particle behaviour at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimise. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic ‘silver bullet’ for improving the quality of every cryoEM sample preparation.

scholarly journals Through-grid wicking enables high-speed cryoEM specimen preparation

2020 ◽  
Author(s):  
Yong Zi Tan ◽  
John L. Rubinstein
Keyword(s):  
Glass Fiber ◽  
High Speed ◽  
Protein Complexes ◽  
Absorption Capacity ◽  
Specimen Preparation ◽  
Water Interface ◽  
Time Resolved ◽  
Glass Fiber Filter ◽  
Liquid Absorption ◽  
Air Water Interface

AbstractBlotting 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 ‘back’ 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 ‘Back-it-up’, 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 for both soluble and detergent-solubilized protein complexes. The device’s speed increases the number of views for a specimen that suffers from preferred orientations.

Enhancement of Contrast in the CEM and STEM Using Bumpy Specimens and Employing the “Dappled Field” Mode of Operation

1974 ◽  
Vol 32 ◽  
pp. 552-553 ◽  
Author(s):  
L. Gandolfi ◽  
J. Reiffel
Keyword(s):  
Operating Mode ◽  
Scanning Transmission Electron Microscope ◽  
Dark Field ◽  
Specimen Preparation ◽  
Field Mode ◽  
Preparation Methods ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Mode Of Operation ◽  
Scanning Transmission

Calculations have been performed on the contrast obtainable, using the Scanning Transmission Electron Microscope, in the observation of thick specimens. Recent research indicates a revival of an earlier interest in the observation of thin specimens with the view of comparing the attainable contrast using both types of specimens.Potential for biological applications of scanning transmission electron microscopy has led to a proliferation of the literature concerning specimen preparation methods and the controversy over “to stain or not to stain” in combination with the use of the dark field operating mode and the same choice of technique using bright field mode of operation has not yet been resolved.

Scanning electron microscopy of human iliac bone by a simple preparation method

1990 ◽  
Vol 48 (3) ◽  
pp. 226-227
Author(s):  
Toshihiko Takita ◽  
Tomonori Naguro ◽  
Toshio Kameie ◽  
Akihiro Iino ◽  
Kichizo Yamamoto
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Preparation Method ◽  
Specimen Preparation ◽  
Real Surface ◽  
Public Attention ◽  
Preparation Methods ◽  
The Real ◽  
Simple Preparation ◽  
Scanning Electron

Recently with the increase in advanced age population, the osteoporosis becomes the object of public attention in the field of orthopedics. The surface topography of the bone by scanning electron microscopy (SEM) is one of the most useful means to study the bone metabolism, that is considered to make clear the mechanism of the osteoporosis. Until today many specimen preparation methods for SEM have been reported. They are roughly classified into two; the anorganic preparation and the simple preparation. The former is suitable for observing mineralization, but has the demerit that the real surface of the bone can not be observed and, moreover, the samples prepared by this method are extremely fragile especially in the case of osteoporosis. On the other hand, the latter has the merit that the real information of the bone surface can be obtained, though it is difficult to recognize the functional situation of the bone.

Microscopy of porous ceramics

1989 ◽  
Vol 47 ◽  
pp. 438-439
Author(s):  
H. M. Kerch ◽  
R. A. Gerhardt
Keyword(s):  
Porous Ceramics ◽  
Specimen Preparation ◽  
High Porosity ◽  
Ion Milling ◽  
Forming Process ◽  
Preparation Methods ◽  
Pore Texture ◽  
Proper Design ◽  
And Function ◽  
Function Information

Highly porous ceramics are employed in a variety of engineering applications due to their unique mechanical, optical, and electrical characteristics. In order to achieve proper design and function, information about the pore structure must be obtained. Parameters of importance include pore size, pore volume, and size distribution, as well as pore texture and geometry. A quantitative determination of these features for high porosity materials by a microscopic technique is usually not done because artifacts introduced by either the sample preparation method or the image forming process of the microscope make interpretation difficult.Scanning electron microscopy for both fractured and polished surfaces has been utilized extensively for examining pore structures. However, there is uncertainty in distinguishing between topography and pores for the fractured specimen and sample pullout obscures the true morphology for samples that are polished. In addition, very small pores (nm range) cannot be resolved in the S.E.M. On the other hand, T.E.M. has better resolution but the specimen preparation methods involved such as powder dispersion, ion milling, and chemical etching may incur problems ranging from preferential widening of pores to partial or complete destruction of the pore network.

Cryo-TEM of amphiphilic polymer and amphiphile/polymer solutions

1993 ◽  
Vol 51 ◽  
pp. 876-877
Author(s):  
Yeshayahu Talmon
Keyword(s):  
Microstructural Characterization ◽  
Building Blocks ◽  
Quantitative Information ◽  
Physical Models ◽  
Specimen Preparation ◽  
Time Resolved ◽  
Temperature Changes ◽  
Temperature And Humidity ◽  
Microstructured Fluids ◽  
Air Streams

To achieve complete microstructural characterization of self-aggregating systems, one needs direct images in addition to quantitative information from non-imaging, e.g., scattering or Theological measurements, techniques. Cryo-TEM enables us to image fluid microstructures at better than one nanometer resolution, with minimal specimen preparation artifacts. Direct images are used to determine the “building blocks” of the fluid microstructure; these are used to build reliable physical models with which quantitative information from techniques such as small-angle x-ray or neutron scattering can be analyzed.To prepare vitrified specimens of microstructured fluids, we have developed the Controlled Environment Vitrification System (CEVS), that enables us to prepare samples under controlled temperature and humidity conditions, thus minimizing microstructural rearrangement due to volatile evaporation or temperature changes. The CEVS may be used to trigger on-the-grid processes to induce formation of new phases, or to study intermediate, transient structures during change of phase (“time-resolved cryo-TEM”). Recently we have developed a new CEVS, where temperature and humidity are controlled by continuous flow of a mixture of humidified and dry air streams.

Methodology for TEM Analysis of Barrier Profiles

2002 ◽  
Author(s):  
Tan-Chen Lee ◽  
Jui-Yen Huang ◽  
Li-Chien Chen ◽  
Ruey-Lian Hwang ◽  
David Su
Keyword(s):  
Sample Preparation ◽  
Profile Analysis ◽  
Image Interpretation ◽  
Sample Thickness ◽  
Image Resolution ◽  
Specimen Preparation ◽  
Tem Analysis ◽  
Preparation Methods ◽  
High Image Resolution ◽  
Common Technique

Abstract Device shrinkage has resulted in thinner barriers and smaller vias. Transmission Electron Microscopy (TEM) has become a common technique for barrier profile analysis because of its high image resolution. TEM sample preparation and image interpretation becomes difficult when the size of the small cylindrical via is close to the TEM sample thickness. Effects of different sample thickness and specimen preparation methods, therefore, have been investigated. An automatic FIB program has been shown to be useful in via sample preparation. Techniques for imaging a TEM specimen will be discussed in the paper. Conventional TEM bright field (BF) image is adequate to examine the barrieronly via; however, other techniques are more suitable for a Cu filled via.

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