Oral Presentation 26th ACMM “2020 Visions in Microscopy”

Cryo-EM grid preparation via SAW atomisation (#71)

Dariush Mr Ashtiani 1 , Adrian Prof Neild 1 , Alex Asso. Prof de Marco 2
  1. Mechanical and Aerospace Engineering, Monash University, Clayton, VIC, Australia
  2. Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia

Cryogenic Transmission Electron Microscopy (cryo-EM) made it possible to easily resolve the structure of proteins and protein complexes at near-atomic resolution. Here, proteins are suspended in an aqueous solution that must be flash-frozen at cryogenic temperature in order to prevent crystalline ice formation. Further, in order to achieve the best imaging conditions, it is critical that the sample thickness is minimized (typically <100 nm). Conventional preparation methods include a blotting step to regulate the amount of solution remaining on the grid, this results in a slow process which cannot be used to time-resolved biochemical reactions and most importantly demonstrated to be incompatible with the preparation of many unstable samples. Recently the idea of “spray and plunge” has demonstrated to execute this process with high efficiency and to be gentler with protein complexes. This approach not only eliminates the hassles associated with the traditional methods but it provides the means for time resolved studies of biochemical reactions which has the potential to revolutionise drug design and discovery. We applied a Surface Acoustic Wave (SAW) atomiser and characterised it as per our application1. These ultrasonic atomisers are easily miniaturisable and can work stand-alone on RF signal without the need for bulky equipment. The sample consumption can be as little as 50 nanolitre and the dead volume is significantly low thanks to our capillary-fed micro-channel bonded on its substrate which can nicely provide steady flow and hence steady aerosol jet even through bursts as short as 50 ms. Our results show that the SAW atomiser can be used to prepare samples for TEM and derive a 3D model of 70S bacterial Ribosome without any major impact on this protein structure2. This system can be retrofitted into the existing conventional plunge freezing devices.

  1. Ashtiani, D.; de Marco, A.; Neild, A., Tailoring surface acoustic wave atomisation for cryo-electron microscopy sample preparation. Lab on a Chip 2019, 19 (8), 1378-1385.
  2. Ashtiani, D.; Venugopal, H.; Belousoff, M.; Spicer, B.; Mak, J.; Neild, A.; de Marco, A., Delivery of femtolitre droplets using surface acoustic wave based atomisation for cryo-EM grid preparation. Journal of structural biology 2018, 203 (2), 94-101