A Novel Live-Cell Microscopy Platform for Real-Time Visualization of 53BP1 Foci Dynamics and Accurate Dosimetry in...

Proton-induced cell death is primarily driven by the induction and repair of DNA double strand breaks. While DNA damage dynamics have been extensively studied, the early cellular responses to proton irradiation remain underexplored. To address this, we developed a novel live-cell microscopy platform that enables real-time visualization of cellular responses to DNA damage induced by proton therapy.

We designed a modular set-up with the requirement that it can be assembled and disassembled within 30 minutes, allowing for efficient deployment in an R&D proton beam line. An inverted fluorescence microscope was mounted at a 90-degree angle relative to the horizontal proton beam, enabling accurate irradiation at various depths along the spread-out Bragg peak with precise dosimetry and control over dose rates. As a proof-of-concept, we investigated the formation of 53BP1 foci following proton irradiation and determined the foci dynamics over time.

With this setup, we observed endogenous 53BP1 foci pre-irradiation, with radiation-induced foci appearing as early as 4 minutes post-irradiation. The maximum number of 53BP1 foci was observed 12 minutes after irradiation, and the foci could be tracked up to 30 minutes post-irradiation.

Our platform enabled precise dosimetry and real-time monitoring of 53BP1-mClover-labeled FaDu cells during proton exposure. This robust setup holds significant potential for studying DNA damage repair dynamics at various positions along the Bragg peak and across different dose rates, including ultrahigh dose rates (FLASH).