Periodic Reporting for period 1 - CryoChip (Nanofluidic chips for reproducible cryo-EM sample preparation with picoliter sample volumes)
Reporting period: 2022-05-01 to 2023-10-31
Cryogenic transmission electron microscopy (cryo-EM) is a technique for high-resolution imaging of biological macromolecules under near-native conditions. In recent years this method has seen dramatic development and has now become the method of choice for structural biology of large macromolecular complexes used by hundreds of academic and industrial research laboratories worldwide. To image and obtain a three-dimensional structure, an aqueous solution containing the protein of interest must be rapidly cooled to form a uniform layer of amorphous ice, ideally not much thicker than the particle itself (~20-100 nm), in which the particles are partitioned in random orientations. The prevailing method for making thin specimens suitable in cryo-EM has not changed substantially since its introduction several decades ago: a filter paper is pressed against one or both sides of a thin, fenestrated support film spanning a metallic mesh grid across which a droplet of the biological sample is suspended, and then rapidly plunged into a liquid cryogen. A recognised shortcoming of this method is that a reproducibly uniform ice layer can often not be achieved. As a result, it may require weeks of optimisation to find conditions suitable for high-resolution imaging, and these conditions are typically different for each sample. Even if such conditions are found, additional problems encountered in practice include preferential orientation of particles, preferential interaction of particles with the support film and denaturation of particles due to exposure to the large air-water interface. This severely limits throughput and impedes any attempts at automation of sample preparation. It is therefore widely appreciated that sample preparation is the major remaining bottleneck precluding cryo-EM from realizing its full potential.
We have developed a novel approach for cryo-EM sample prepraration using a Micro-Electromechanical-Systems (MEMS)-based nanofluidic chip with a free-standing electron transparent observation window that can be filled with protein solution. In our design, specimen thickness reproducibly constrained by the dimensions of the nanofluidic channel. In addition, the destructive effect of the air-water interface on macromolecules is entirely avoided. We expect this approach to be capable of mitigating many of the challenges that are limiting present cryo-EM sample preparation methods: It requires only picoliter sample volumes, it can robustly provide uniform thickness gradients across the sampling area without the need for any optimisation or expensive instrumentation, which together will allow complete automation of the sample preparation process.
We have developed a novel approach for cryo-EM sample prepraration using a Micro-Electromechanical-Systems (MEMS)-based nanofluidic chip with a free-standing electron transparent observation window that can be filled with protein solution. In our design, specimen thickness reproducibly constrained by the dimensions of the nanofluidic channel. In addition, the destructive effect of the air-water interface on macromolecules is entirely avoided. We expect this approach to be capable of mitigating many of the challenges that are limiting present cryo-EM sample preparation methods: It requires only picoliter sample volumes, it can robustly provide uniform thickness gradients across the sampling area without the need for any optimisation or expensive instrumentation, which together will allow complete automation of the sample preparation process.
Compared to the original protoype, we have increased the number of imaging membranes to 36, with a total of ~20,000 images in homogeneously thin amorphous ice. This is significantly more than we had originally anticipated for the CryoChip design for this proposal. We were able to realise this through improved transversal patterning of mechanical stabilisation elements in the MEMS design. We also improved the filling capacity and the vitrification efficiency through modifications of the chip base design. Using our standard set of test proteins we have verified we can routinely use high-resolution 3D reconstructions for these samples.
We have designed and manufactured an improved plunging system for robust filling and vitrification of CryoChips and have distributed these for field testing to the cryoEM facilities of the user consortium. Data from application of these devices from user experiments is being collected for analysis in collaboration with the facilities. Together with our industrial partner, we have scaled up production of the CryoChip prototype for dissemination and field testing at the cooperating facilities. CryoChips have been shipped to collaborating facilities for field testing on a broad range of cryoEM samples. Analysis of accumulated data for testing parameters (efficiency of sample application, mechanical stability, reproducibility and uniformity of ice thickness, beam-induced motion, obtainable resolution, particle orientation assessment and automation of data collection) will be analysed once the facilities have finalised the field testing phase.
We have designed and manufactured an improved plunging system for robust filling and vitrification of CryoChips and have distributed these for field testing to the cryoEM facilities of the user consortium. Data from application of these devices from user experiments is being collected for analysis in collaboration with the facilities. Together with our industrial partner, we have scaled up production of the CryoChip prototype for dissemination and field testing at the cooperating facilities. CryoChips have been shipped to collaborating facilities for field testing on a broad range of cryoEM samples. Analysis of accumulated data for testing parameters (efficiency of sample application, mechanical stability, reproducibility and uniformity of ice thickness, beam-induced motion, obtainable resolution, particle orientation assessment and automation of data collection) will be analysed once the facilities have finalised the field testing phase.
We have realised a robust MEMS-based sample support carrier for cryo-EM featuring nanofluidic cavities. We have successfully scaled production to supply our international user consortium with ample material for field testing. Our new design shows improved filling capacity, robust vitrification properties and high imaging efficiency. A dedicated and semi-automated filling and vitrification station also significantly facilitates sample handling. Further research investigating possible surface passivation strategies is required to develop the prototype into a mature MVP. Analysis of the field testing performance using data acquired by our collaborating user consortium will form the basis for additional design cycles on the way to a MVP.