Tracks the fate of one of these cloud-like foci through time in seconds; fast reappearance of Green signal after photobleaching indicates fast exchange of the protein—the surprise is the subcomparments within each of those foci which was not recognized previously). Credit: Queen Mary, University of London
Scientists at the Center for Cell Dynamics, School of Biological and Behavioral Sciences, Queen Mary University of London, in collaboration with Carl Zeiss, have developed an innovative live-cell imaging technique that combines an exceptional resolution of 60 nanometers with fluorescence recovery after photobleaching, while significantly reducing light-induced cellular damage. This advancement allows researchers to observe intricate cellular processes with unprecedented clarity, opening new avenues for understanding fundamental biological mechanisms, including DNA repair and chromosome dynamics. The technology can also facilitate novel live-cell dynamics based drug target and drug screening methods that transcend the diffraction limit of systems.
The team, led by Professor Viji Draviam, has ingeniously combined Lattice Structured Illumination Microscopy (diSIM/SIM²) with Fluorescence Recovery After Photobleaching (FRAP) to create a novel method termed FRAP-SR (FRAP in Super-Resolution regime). This technique overcomes the limitations of traditional microscopy and earlier super-resolution methods, which often suffer from phototoxicity, hindering the study of delicate biological events in living cells. The findings are published on the bioRxiv preprint server.
"Our FRAP-SR approach enables us to visualize structures as small as 60 nanometers within living cells—a scale previously inaccessible for dynamic studies without causing significant cellular stress," explains Professor Draviam. "This resolution, 2000 times smaller than the width of a human hair, allows us to probe the nanoscale organization and behavior of cellular components in real-time."
Using FRAP-SR, the researchers investigated the dynamics of 53BP1, a key protein involved in the repair of double-strand DNA breaks. Their high-resolution live-cell imaging revealed that 53BP1 forms liquid-like condensates with surprising complexity. Some of these foci appeared as stable, compact structures, while others exhibited more fluid, dynamic shapes.
Full nuclei with DNA damage spots decorated by green cloud-like 53BP1-foci. Credit: Queen Mary, University of London
By applying FRAP-SR, the team discovered that the amorphous 53BP1 foci contain distinct subcompartments with varying protein mobility, suggesting functional specialization within these repair centers. In contrast, compact foci displayed uniform recovery after photobleaching but showed greater heterogeneity in recovery rates between different foci. The study also revealed that the dynamics of these foci are influenced by cellular conditions, such as recovery from DNA replication stress.
Professor Draviam highlights the potential of this innovation: "FRAP-SR provides a powerful tool to dissect the dynamic architecture of protein assemblies at the nanoscale in living cells. It allows us to investigate fundamental cellular processes, particularly those sensitive to light exposure, with unprecedented detail and minimal perturbation. This will transform the field of optogenetics in the super-resolution regime. It will also enable the development of new anti-cancer drugs that target DNA damage repair pathways that are dynamic"
This advancement holds significant promise for cell biology researchers studying a wide range of light-sensitive processes, including DNA damage response, chromosome organization, mitochondrial dynamics, and cellular senescence. The ability to study these processes at high resolution in living cells without inducing damage will undoubtedly accelerate discoveries in these fields.
The global DNA repair drugs market was valued at approximately USD 9.18 billion in 2024 and is projected to reach USD 13.97 billion by 2030, growing at a compound annual growth rate (CAGR) of around 7.2%. The team has shown how the DNA damage marker 53BP1 can be exploited in live-cells using FRAP-SR to accelerate the development of novel DNA repair drugs or drug candidates relevant to personalized medicine.
The ZEISS Elyra 7 system, enhanced with FRAP capabilities from Rapp OptoElectronics, was instrumental in this study, providing the advanced super-resolution imaging necessary to resolve the subcompartments of 53BP1 foci for the first time. Professor Draviam's collaboration with Zeiss and Rapp OptoElectronics to integrate FRAP and structured illumination microscopy allowed for precise quantification of protein dynamics.
