Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation

Ashtiani, Dariush; Venugopal, Hari; Belousoff, Matthew J.; Spicer, Bradley A.; Mak, Johnson; Neild, Adrian; Marco, Alex de

doi:10.1016/j.jsb.2018.03.012

Cited by 38 publications

(28 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the experiments described above, we used the same ultrasonic specimen-application approach as employed with the Shake-it-off instrument, which is related to an acoustic wave atomization approach described previously (Ashtiani et al, 2018). However, through-grid wicking can almost certainly be used with other methods for applying sample onto an EM grid.…”

Section: Discussionmentioning

confidence: 99%

Through-grid wicking enables high-speed cryoEM specimen preparation

Tan

Rubinstein

2020

Acta Crystallogr D Struct Biol

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Through-grid wicking enables high-speed cryoEM specimen preparation

Tan

Rubinstein

2020

Acta Crystallogr D Struct Biol

View full text Add to dashboard Cite

show abstract

“…Smaller nanoliter-sized droplets have also been generated from microfluidic devices using surface acoustic waves (SAW) [164]. The same technique has been used to deliver femtoliter-sized droplets for cryo-EM grid preparation [165]. Drop-on-demand therefore has great potentials in rivalling jet-based methods in cutting sample consumption.…”

Section: Drop-on-demand-potential To Maximize Sample Efficiencymentioning

confidence: 99%

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Cheng¹

2020

Crystals

View full text Add to dashboard Cite

The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.

show abstract

“…To improve sample deposition on EM grids, several developments have been presented, including droplet-based methods [20][21][22][23][24] , e.g. on nanowire grids 25 , and using capillaries followed by sample thinning by controlled water evaporation 26 .…”

Section: Pin Printingmentioning

confidence: 99%

Automated cryo-EM sample preparation by pin-printing and jet vitrification

Ravelli

Nijpels

Henderikx

et al. 2019

Preprint

View full text Add to dashboard Cite

The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several technical developments that provide controlled and efficient sample preparation for cryo-EM studies.Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving 4 standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.

show abstract

Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation

Cited by 38 publications

References 48 publications

Through-grid wicking enables high-speed cryoEM specimen preparation

Through-grid wicking enables high-speed cryoEM specimen preparation

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Automated cryo-EM sample preparation by pin-printing and jet vitrification

Contact Info

Product

Resources

About