Periodic Reporting for period 1 - RT-SuperES (Real-Time high-content Super-Resolution Imaging of ES Cell States)
Reporting period: 2023-07-01 to 2024-06-30
RT-SuperES aims to revolutionise the field of biomedical research and industry by developing a cutting-edge real-time super-resolution (SR) microscope with unparalleled spatial and temporal resolution. This innovative technology will enable nanoscale observations of cellular behaviour, particularly in embryonic stem cells, thereby addressing a significant need within the scientific community. The project's objective is to enhance the competitiveness of the European robotic-based microscopy industry and establish a new premium market for advanced SR technology.
The biomedical research community currently faces limitations in extracting and integrating detailed molecular features and dynamics from individual cells within a population. These constraints hinder a comprehensive understanding of how cells transition into different states and fates. Moreover, there is a lack of efficient technical implementations of smart microscopy that can be widely adopted. The RT-SuperES project seeks to bridge this gap by developing a Machine Learning (ML)-based control system for high-content SR microscopes, enabling real-time observations and analysis. The consortium includes experts in stem cell biology, nuclear dynamics, super-resolution imaging, artificial intelligence, and advanced microscopy. The project aims to break new ground by combining these diverse fields and successfully complete the RT-SuperES instrument.
RT-SuperES expected impacts are:
1. **Technological Advancements**: The project will pioneer a new solution for microscopy, integrating machine learning to control high-content SR microscopes. This advancement will extend the developed concepts to other microscopy techniques, significantly impacting the biomedical research community by providing more efficient and versatile tools.
2. **Biomedical Research**: The new SR ML-driven imaging instrument will enable tracking the dynamics of relevant features involved in cell fate transitions. The project will also result in a library of endogenously Halo-tagged proteins in embryonic stem cells, freely available to academia and industry either as single clones or as en entire library. This resource will facilitate a deeper understanding of molecular and cellular processes.
3. **Drug Development**: The methodologies developed should greatly interest the biotechnology and pharmaceutical industries. The commercial imaging systems and novel assays for drug screening produced by RT-SuperES will aid in developing new drugs, improving the efficiency and effectiveness of drug discovery processes.
In the political and strategic context, RT-SuperES aligns with European strategic objectives to foster innovation and enhance industrial competitiveness. It opens up new markets and research opportunities by placing the European industry at the forefront of SR microscopy technology. RT-SuperES will contribute significantly to the scientific community and the broader biomedical and pharmaceutical industries, addressing both existing needs and creating new possibilities.
Scale and significance of impact: RT-SuperES is anticipated to have a broad and profound impact on multiple fields. The technological advancements will set a new standard in microscopy, potentially influencing research methodologies worldwide. The contributions to biomedical research and drug development will accelerate scientific discoveries and the creation of new treatments, ultimately benefiting global health. The project will stimulate economic growth and innovation by establishing Europe as a leader in advanced SR microscopy, reinforcing the region's strategic position in the global market.
This comprehensive approach sets the stage for RT-SuperES, highlighting its potential to drive significant technological advancements, research, and industry, addressing critical needs and fostering innovation across multiple sectors.
The biomedical research community currently faces limitations in extracting and integrating detailed molecular features and dynamics from individual cells within a population. These constraints hinder a comprehensive understanding of how cells transition into different states and fates. Moreover, there is a lack of efficient technical implementations of smart microscopy that can be widely adopted. The RT-SuperES project seeks to bridge this gap by developing a Machine Learning (ML)-based control system for high-content SR microscopes, enabling real-time observations and analysis. The consortium includes experts in stem cell biology, nuclear dynamics, super-resolution imaging, artificial intelligence, and advanced microscopy. The project aims to break new ground by combining these diverse fields and successfully complete the RT-SuperES instrument.
RT-SuperES expected impacts are:
1. **Technological Advancements**: The project will pioneer a new solution for microscopy, integrating machine learning to control high-content SR microscopes. This advancement will extend the developed concepts to other microscopy techniques, significantly impacting the biomedical research community by providing more efficient and versatile tools.
2. **Biomedical Research**: The new SR ML-driven imaging instrument will enable tracking the dynamics of relevant features involved in cell fate transitions. The project will also result in a library of endogenously Halo-tagged proteins in embryonic stem cells, freely available to academia and industry either as single clones or as en entire library. This resource will facilitate a deeper understanding of molecular and cellular processes.
3. **Drug Development**: The methodologies developed should greatly interest the biotechnology and pharmaceutical industries. The commercial imaging systems and novel assays for drug screening produced by RT-SuperES will aid in developing new drugs, improving the efficiency and effectiveness of drug discovery processes.
In the political and strategic context, RT-SuperES aligns with European strategic objectives to foster innovation and enhance industrial competitiveness. It opens up new markets and research opportunities by placing the European industry at the forefront of SR microscopy technology. RT-SuperES will contribute significantly to the scientific community and the broader biomedical and pharmaceutical industries, addressing both existing needs and creating new possibilities.
Scale and significance of impact: RT-SuperES is anticipated to have a broad and profound impact on multiple fields. The technological advancements will set a new standard in microscopy, potentially influencing research methodologies worldwide. The contributions to biomedical research and drug development will accelerate scientific discoveries and the creation of new treatments, ultimately benefiting global health. The project will stimulate economic growth and innovation by establishing Europe as a leader in advanced SR microscopy, reinforcing the region's strategic position in the global market.
This comprehensive approach sets the stage for RT-SuperES, highlighting its potential to drive significant technological advancements, research, and industry, addressing critical needs and fostering innovation across multiple sectors.
In WP1, we designed three plasmids to create an endogenous mouse embryonic stem cell library, each capable of infecting cells due to their viral properties and containing antibiotic resistance for cells and bacteria. These plasmids include a HA-tag for chromatin immunoprecipitation sequencing (ChIP-seq) and a Halo-tag sequence without start or stop codons to ensure expression only when the integrated gene is active. They also feature 5' and 3' splice sites, allowing the Halo-tag to be treated as a novel exon when integrated into a gene. The only variation among the plasmids is the addition of three nucleotides to account for different reading frame shifts. Concurrently, we began karyotyping embryonic stem cells to ensure chromosomal integrity, examining two cell lines to use a genetically normal line. Additionally, we generated three Halo-tag knock-in lines using CRISPR/Cas9, tagging OCT4, NANOG, and β-Catenin, all of which showed correct localisation and expression, supporting super-resolution imaging.
In WP4, a digital micro-mirror device (DMD)-based structured excitation was implemented with localisation microscopy, achieving a gain of 2 in resolution. Depending on the single molecule approach (PALM/STORM/DNA-PAINT), higher power lasers may be needed to reach the necessary irradiance levels. The DMD was also used to implement the mSIM approach by creating and scanning a point matrix on the sample, then processing the images with pixel reassignment to increase resolution, with typical acquisition times around 1 second. This setup is highly flexible, allowing full tuning of the excitation matrix parameters, and supports both single molecule localisation microscopy and mSIM. Further development of this system is planned over the next three years.
In WP4, a digital micro-mirror device (DMD)-based structured excitation was implemented with localisation microscopy, achieving a gain of 2 in resolution. Depending on the single molecule approach (PALM/STORM/DNA-PAINT), higher power lasers may be needed to reach the necessary irradiance levels. The DMD was also used to implement the mSIM approach by creating and scanning a point matrix on the sample, then processing the images with pixel reassignment to increase resolution, with typical acquisition times around 1 second. This setup is highly flexible, allowing full tuning of the excitation matrix parameters, and supports both single molecule localisation microscopy and mSIM. Further development of this system is planned over the next three years.