Gentle live-cell imaging at super resolution

Scientists at Queen Mary University of London, in collaboration with optical technology company Carl Zeiss, have developed an innovative live-cell imaging technique that combines an impressive resolution of 60 nm with fluorescence recovery after photobleaching, while significantly reducing light-induced cellular damage.

Described in the journal Cell Reports Methods, the new method is said to enable the observation of 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 research team, led by Professor Viji Draviam, 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.

“Our FRAP-SR approach enables us to visualise structures as small as 60 nm within living cells — a scale previously inaccessible for dynamic studies without causing significant cellular stress,” Draviam said. “This resolution, 2000 times smaller than the width of a human hair, allows us to probe the nanoscale organisation and behaviour 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.

By applying FRAP-SR, the team discovered that the amorphous 53BP1 foci contain distinct subcompartments with varying protein mobility, suggesting functional specialisation within these repair centres. 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.

The ZEISS Elyra 7 system, enhanced with FRAP capabilities from Rapp OptoElectronic, provided the advanced super-resolution imaging necessary to resolve the subcompartments of the 53BP1 foci. Draviam’s collaboration with Zeiss and Rapp OptoElectronics to integrate FRAP and structured illumination microscopy allowed for precise quantification of protein dynamics.

“FRAP-SR provides a powerful tool to dissect the dynamic architecture of protein assemblies at the nanoscale in living cells,” Draviam said. “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 anticancer 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 organisation, 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.