Innovative method of superimposing Gamma-CBCT images with live confocal imaging to assess Cellular Responses to Radiation (Preprint)

<sec> <title>BACKGROUND</title> Understanding the real-time effects of radiation on live cells remains a challenge due to limitations in current imaging and targeting techniques. Traditional methods like clonogenic assays and fixed-cell imaging fail to capture dynamic and spatially-resolved cellular responses. </sec> <sec> <title>OBJECTIVE</title> This study introduces and evaluates three novel phantom-based methodologies designed to visualize and analyze radiation-induced changes in live cells with high spatial precision. </sec> <sec> <title>METHODS</title> All three techniques employed confocal imaging dishes embedded in brain-mimicking agarose gel phantoms. Live cells were cultured on treated glass-bottom dishes and exposed to targeted gamma radiation. Technique 1 involved direct embedding of the dish without any positional markers. Technique 2 incorporated a small metal grid and aperture film to partially localize radiation effects. Technique 3 employed a full-scale metal grid for precise spatial mapping and dose-effect correlation. Cone-beam CT (CBCT) was used for localization, and post-radiation cellular effects were assessed using confocal microscopy. </sec> <sec> <title>RESULTS</title> Technique 1 allowed basic observation of radiation effects but lacked spatial localization. Technique 2 enabled partial localization via CBCT, yet limitations in grid size and imaging overlay reduced resolution. Technique 3 demonstrated superior precision, enabling clear mapping of dose-dependent cellular responses in individual grid sections. However, challenges such as metal-media interaction and grid handling remained. </sec> <sec> <title>CONCLUSIONS</title> The full-scale metal grid approach (Technique 3) offers a powerful platform for studying real-time radiation effects on live cells with high spatial resolution. These findings open new avenues in radiobiology, particularly in optimizing radiotherapy protocols and investigating radiation damage mechanisms. Further refinements, including grid material optimization and integration with advanced imaging modalities, are warranted. </sec>