Three-dimensional fluorescence and intensity diffraction tomography dual-modality imaging based on adaptive optics

Intensity diffraction tomography (IDT) and fluorescence dual-modality imaging techniques can provide comprehensive analysis of biological components and their interactions. However, long-term imaging is usually affected by environmental temperature fluctuations and mechanical disturbances of the microscope, leading to time-varying aberrations and focus drift, which in turn degrade the performance of long-term imaging and the accuracy of dual-modality colocalization. To address these issues, we propose three-dimensional (3D) fluorescence and intensity diffraction tomography dual-modality imaging based on adaptive optics. This method employs an innovative iterative structured diffraction tomography imaging approach combined with annular matching illumination conditions to precisely reconstruct the IDT results and characterize aberrations in real time. Moreover, we feedback the real-time aberrations to the system’s point spread function to synchronously correct the 3D fluorescence imaging results. By conducting a detailed analysis of the morphological features of subcellular organelles in live COS-7 cells, we demonstrate the broad application prospects of the 3D dual-modality imaging method with adaptive optics in the analysis of subcellular organelle structure and function.