Adaptive Optics‐Assisted Long‐Term 3D Fluorescence and Intensity Diffraction Tomography for High Spatiotemporal Resolution Cellular Imaging

Abstract Intensity diffraction tomography (IDT) and fluorescence dual‐modality imaging facilitate a comprehensive analysis of biological components and their interactions. However, long‐term imaging is typically compromised by environmental thermal fluctuations and mechanical disturbances from the microscope, leading to time‐varying aberrations and focus drift that degrades the performance of long‐term imaging and the accuracy of dual‐modality colocalization. To counter these issues, an adaptive optics‐assisted 3D dual‐modality imaging method (AO‐FIDT) is developed. The method utilizes an innovative iterative ptychographic approach, paired with annular matched illumination conditions, to precisely reconstruct IDT results and characterize the aberrations in real‐time. Furthermore, feedback on the real‐time aberrations to the point spread function of the system is provided to synchronously correct the 3D fluorescence results. The efficacy and precision of AO‐FIDT are confirmed through long‐term, high‐resolution imaging of HeLa cells. Furthermore, by scrutinizing the morphological characteristics of subcellular organelles in live COS‐7 cells, including progressive sphericity in mitochondria under phototoxicity, and by monitoring the continuous changes in mitochondrial dynamics throughout the cell division process, the broad applicability of AO‐FIDT in analyzing subcellular organelle structure and function is demonstrated.