Live Cell Superresolution Method Enables Full Range of Biological Mechanism Imaging

LONDON, Aug. 18, 2025 — Researchers introduced a superresolution imaging technique that visualizes live, dynamic cellular structures at 60-100 nm while significantly reducing the risk of damaging the fragile cells. The microscopy advancement could inform research into DNA repair, chromosome activity, and other biological mechanisms.

The imaging approach, developed by a team at Queen Mary University of London in collaboration with industry partners, combines Fluorescence Recovery After Photobleaching (FRAP) with Lattice Structured Illumination Microscopy (diSIM/SIM²). The resulting application is named FRAP in the Superresolution regime (FRAP-SR).

Full nuclei exhibiting DNA damage, marked by green, cloud-like 53BP1 foci. Courtesy of Queen Mary University of London.

“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,” professor Viji Draviam, who led the research, said. “This resolution, 2000x 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 a protein that is key to the repair of double-strand DNA breaks, called 53BP1. They analyzed the dynamics of the 53BP1 protein within nuclear structures at 60-nm resolution. FRAP-SR enabled them to correlate protein diffusion with subcellular structural changes in the superresolution regime without perturbing the live-cell samples.

The approach revealed sub-compartments within 53BP1 foci. These sub-compartments displayed faster 53BP1 protein mobility than other foci without sub-compartments.

The researchers characterized two distinct types of 53BP1 foci that differed in their activities. Some foci appeared as stable, compact structures, while others exhibited more fluid, dynamic shapes. The compact foci displayed uniform recovery after photobleaching, but showed greater heterogeneity in the recovery rates between different foci. The amorphous foci contained discrete sub-compartments with varying protein mobility, suggesting functional specialization within these DNA repair centers.

Using lattice light-sheet movies of aphidicolin-treated cells, the researchers confirmed faster recovery of 53BP1 in amorphous foci compared to compact foci. The study also revealed that the dynamics of the foci are influenced by cellular conditions such as recovery from DNA replication stress.

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The team believes that, over the long term, the combination of FRAP and diSIM will enable scientists to overcome existing limitations to examining photosensitive subcellular structures with varied protein mobilities, activities, and roles. Long-range joining of DNA breaks are important, as defective cells can experience extensive degradation of the unrepaired coding ends, leading to genomic instability.

FRAP-SR tracks foci through time in seconds. The fast reappearance of the green signal after photobleaching indicates fast exchange of the protein. The sub-compartments within each foci were not previously recognized. Courtesy of Queen Mary University of London.

FRAP-SR could accelerate the development of drug targeting and drug screening methods based on live-cell dynamics. The global market for DNA repair drugs was valued at approximately $9.18 billion in 2024 and is projected to reach $13.97 billion by 2030. The use of FRAP-SR to study the DNA damage marker, 53BP1, in live cells could foster the development of DNA repair drugs and candidate drugs for personalized medicine.

“This will transform the field of optogenetics in the superresolution regime," Draviam said. "It will also enable the development of new anti-cancer drugs that target DNA damage repair pathways that are dynamic.”

The study utilized the ZEISS Elyra 7 system, enhanced with FRAP capabilities from Rapp OptoElectronics. The system provided the superresolution imaging necessary to resolve the sub-compartments of 53BP1 foci. The researchers worked with ZEISS and Rapp OptoElectronics to integrate FRAP and SIM, which allowed for precise quantification of protein dynamics.

The research was published in Cell Reports Methods (www.doi.org/10.1101/2025.05.07.652606).

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Published: August 2025

Glossary

structured illumination microscopy: Structured illumination microscopy (SIM) is an advanced optical imaging technique used in microscopy to enhance the resolution of images beyond the diffraction limit imposed by traditional light microscopy. The diffraction limit is a fundamental limitation that restricts the ability to distinguish fine details in the microscopic structures. SIM achieves improved resolution through a process of illuminating the specimen with a patterned light, typically a grid or a stripe pattern. This...
superresolution: Superresolution refers to the enhancement or improvement of the spatial resolution beyond the conventional limits imposed by the diffraction of light. In the context of imaging, it is a set of techniques and algorithms that aim to achieve higher resolution images than what is traditionally possible using standard imaging systems. In conventional optical microscopy, the resolution is limited by the diffraction of light, a phenomenon described by Ernst Abbe's diffraction limit. This limit sets a...
optogenetics: A discipline that combines optics and genetics to enable the use of light to stimulate and control cells in living tissue, typically neurons, which have been genetically modified to respond to light. Only the cells that have been modified to include light-sensitive proteins will be under control of the light. The ability to selectively target cells gives researchers precise control. Using light to control the excitation, inhibition and signaling pathways of specific cells or groups of...
nano: An SI prefix meaning one billionth (10-9). Nano can also be used to indicate the study of atoms, molecules and other structures and particles on the nanometer scale. Nano-optics (also referred to as nanophotonics), for example, is the study of how light and light-matter interactions behave on the nanometer scale. See nanophotonics.
fluorescence: Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
photobleaching: Photobleaching is a phenomenon in which the fluorescence of a fluorophore (a fluorescent molecule or dye) is permanently reduced or eliminated upon prolonged exposure to light. This process occurs due to the photochemical destruction or alteration of the fluorophore molecules, rendering them non-fluorescent. Key points about photobleaching: Mechanism: Photobleaching is typically a result of chemical reactions induced by the absorbed photons. The excessive light exposure causes the...

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