Periodic Reporting for period 3 - Cryo-SECOM Workflow (Unlocking the potential of cryo-electron tomography by simplification of the sample preparation workflow )
Reporting period: 2022-09-01 to 2023-05-31
With the advent of direct electron detectors, the Cryo-Electron Microscopy (cryo-EM) and Cryo-Electron Tomography (cryo-ET) showed a spectacular growth trend as a widely adopted technology in molecular and structural biology, resolving protein structures and unveiling their interactions in relevant biological specimens, such as cells or bacteria. Since the Nobel Prize in Chemistry in 2017, the number of protein structure depositions in the EM Data Bank (EMDB) has more than doubled. However, the best example illustrating this trend nowadays is related to the global Covid-19 crisis - the number of publications reporting cryo-EM data related only to the SARS-CoV-2 virus alone has exceeded 13.000 since the outbreak in 2019.
Despite increasingly growing availability, Life Science researchers and industry are yet to fully benefit from the cryo-ET technology, since the sample preparation process remains a major bottleneck in the workflow. The common process success rate is less than 20% due to the 22 steps, including multiple transfers, that increase the risk of sample contamination and low reproducibility. The process takes 4-8 hours for an experienced PhD level operator and often has to be fully repeated due to the limited inter-step feedback before transferring the sample into a cryo-TEM. Thus, sample preparation is also incredibly costly, as a large amount of the expensive cryo-TEM imaging time is wasted on sample evaluation. To make cryo-ET fulfil its potential, the sample preparation has to become a fast, cheap, and reliable process, providing researchers with clear feedback about sample quality before the cryo-TEM characterization.
DELMIC has reimagined the cryo-ET workflow, with the ultimate vision of a completely automated sample preparation carried out in a single workstation – the Cryo-SECOM, which integrates a correlated light microscope with a cryo-FIB system. Additionally, actively cooled & vacuum-sealed transfer compatible with Cryo-SECOM and cryo-TEM autoloaders facilitate sample handling and prevent contamination. This fully integrated workflow approach offers high precision in targeting the area of interest and aims to improve a cryo-lamella yield by 8-fold, with nearly 90% reduced cost and preparation time. The Cryo-SECOM Workflow will provide researchers with a highly-reproducible methodology, triggering fast-paced innovation in structural biology research by unlocking the full potential of cryo-ET cost-effectively.
Despite increasingly growing availability, Life Science researchers and industry are yet to fully benefit from the cryo-ET technology, since the sample preparation process remains a major bottleneck in the workflow. The common process success rate is less than 20% due to the 22 steps, including multiple transfers, that increase the risk of sample contamination and low reproducibility. The process takes 4-8 hours for an experienced PhD level operator and often has to be fully repeated due to the limited inter-step feedback before transferring the sample into a cryo-TEM. Thus, sample preparation is also incredibly costly, as a large amount of the expensive cryo-TEM imaging time is wasted on sample evaluation. To make cryo-ET fulfil its potential, the sample preparation has to become a fast, cheap, and reliable process, providing researchers with clear feedback about sample quality before the cryo-TEM characterization.
DELMIC has reimagined the cryo-ET workflow, with the ultimate vision of a completely automated sample preparation carried out in a single workstation – the Cryo-SECOM, which integrates a correlated light microscope with a cryo-FIB system. Additionally, actively cooled & vacuum-sealed transfer compatible with Cryo-SECOM and cryo-TEM autoloaders facilitate sample handling and prevent contamination. This fully integrated workflow approach offers high precision in targeting the area of interest and aims to improve a cryo-lamella yield by 8-fold, with nearly 90% reduced cost and preparation time. The Cryo-SECOM Workflow will provide researchers with a highly-reproducible methodology, triggering fast-paced innovation in structural biology research by unlocking the full potential of cryo-ET cost-effectively.
During the initial phase of the Project, DELMIC built a retrofit Cryo-SECOM prototype complying with the TRL7 requirements. At that stage, we proved that our technology can produce FLM-guided cryo-lamellas free of contaminants and suitable for cryo-ET tomogram acquisition.
After obtaining these initial results, our efforts were focused on development of the standalone prototype. Specifically, we pursued the interfacing of the Cryo-SECOM core modules with a dedicated FIB chamber, and development of a dedicated hardware for contamination-free sample preparation and transfer. We also implemented a robust and relatively cheap cryogenic cooler and finalized software development, processing automation, and integration with the system.
In the final phase, we have adapted the optical microscope module to the final Cryo-SECOM FIB chamber. This activity resulted in a fully integrated Cryo-SECOM module, and the release of a side product METEOR - the first commercial add-on FLM microscope on the FIB/SEM market.
Our collaboration with the prototype validation partners led to the development of additional parts of the Workflow - CERES Clean Station, and CERES Ice Shield. Both were commercialized as side products, which prevent sample contamination during the initial preparation step, and further boost sample purity inside the Cryo-SECOM (MIMAS) chamber. Specifically, the presence of the Cold Shield significantly extended the time over which a batch of 8 samples can be processed without risking parasitic ice growth. This opened the possibility for an automated FLM-guided cryo-FIB milling over 24/7.
The external validation of the Cryo-SECOM was completed at the end of the Project. The extensive testing in a real laboratory conditions resulted in a valuable feedback which helped to iterate and freeze the final design of the system.
Other efforts were focused on generating dissemination materials, final commercialization planning and market analysis, as well as securing the post project financing.
After obtaining these initial results, our efforts were focused on development of the standalone prototype. Specifically, we pursued the interfacing of the Cryo-SECOM core modules with a dedicated FIB chamber, and development of a dedicated hardware for contamination-free sample preparation and transfer. We also implemented a robust and relatively cheap cryogenic cooler and finalized software development, processing automation, and integration with the system.
In the final phase, we have adapted the optical microscope module to the final Cryo-SECOM FIB chamber. This activity resulted in a fully integrated Cryo-SECOM module, and the release of a side product METEOR - the first commercial add-on FLM microscope on the FIB/SEM market.
Our collaboration with the prototype validation partners led to the development of additional parts of the Workflow - CERES Clean Station, and CERES Ice Shield. Both were commercialized as side products, which prevent sample contamination during the initial preparation step, and further boost sample purity inside the Cryo-SECOM (MIMAS) chamber. Specifically, the presence of the Cold Shield significantly extended the time over which a batch of 8 samples can be processed without risking parasitic ice growth. This opened the possibility for an automated FLM-guided cryo-FIB milling over 24/7.
The external validation of the Cryo-SECOM was completed at the end of the Project. The extensive testing in a real laboratory conditions resulted in a valuable feedback which helped to iterate and freeze the final design of the system.
Other efforts were focused on generating dissemination materials, final commercialization planning and market analysis, as well as securing the post project financing.
The Cryo-SECOM Workflow is currently one of the three products that cover the majority of steps of the conventional cryo-lamella preparation process in a single workstation. While the competitive systems are strongly focused on the SEM/FIB coexistence being supported by a simple FLM add-on, the MIMAS system has been designed with a high-end FLM/FIB configuration utilising our state-of-the-art METEOR optical microscope. In our system, the FLM microscope plays a key role in localising molecular specimens of interest within the sample volume, as the longitudinal image resolution goes beyond the diffraction limit reaching ~50 nm, even at low signal-to-noise ratios. This directly translates into a higher success rate of the lamella milling process, as the system is much more precise in localising the targeted molecular interactions, ensuring that the lamella is being milled around and not through them. Additionally, we use a special optical interferometric mode to measure a lamella thickness with a ±5 nm precision, thus eliminating the need for an expensive SEM column.
The introduced concept of the sample transfer overcomes the cons of an automated transfer autoloader. Manually operated actively-cooled high-vacuum transfer of a multiple sample batch is now loaded directly on the microscope sample stage. This eliminates moving samples one by one within different microscope modules, reduces the processing time, and improves the sample quality and success rate by ensuring the same milling angle for each grid. Most importantly, our preparation station and cryo shielding prevent parasitic ice growth, thus all the samples remain contamination-free 24/7 until the automatically performed lamella preparation process is completed for the whole batch. With the new approach, the end user gains a system, where a batch of samples is manually placed in the microscope, which is similarly done in the competitive systems, however, we do not use a complicated and expensive autoloader.
The updated Cryo-SECOM concept has several key advantages: (i) state-of-the-art FLM with super-resolution and lamella thickness control capability, making the SEM obsolete, (ii) automated lamella milling process of a whole batch without the need for an expensive autoloader, yet with a high milling angle repeatability (iii) low contamination rates, and >90% success rate for a significantly lower offering price.
The introduced concept of the sample transfer overcomes the cons of an automated transfer autoloader. Manually operated actively-cooled high-vacuum transfer of a multiple sample batch is now loaded directly on the microscope sample stage. This eliminates moving samples one by one within different microscope modules, reduces the processing time, and improves the sample quality and success rate by ensuring the same milling angle for each grid. Most importantly, our preparation station and cryo shielding prevent parasitic ice growth, thus all the samples remain contamination-free 24/7 until the automatically performed lamella preparation process is completed for the whole batch. With the new approach, the end user gains a system, where a batch of samples is manually placed in the microscope, which is similarly done in the competitive systems, however, we do not use a complicated and expensive autoloader.
The updated Cryo-SECOM concept has several key advantages: (i) state-of-the-art FLM with super-resolution and lamella thickness control capability, making the SEM obsolete, (ii) automated lamella milling process of a whole batch without the need for an expensive autoloader, yet with a high milling angle repeatability (iii) low contamination rates, and >90% success rate for a significantly lower offering price.