High-Speed Multi-Modal Extended Depth-of-Field Microscopy with an Electrically Tunable Lens

High spatial resolution imaging over the entire volume for intravital imaging of biological specimens has long been vital. However, the depth-of-field (DOF) and spatial resolution are intrinsically interdependent. Here, a new extended DOF (EDOF) microscopy technique based on partially coherent annular illumination is proposed, termed AI-EDOF, by combining an electrically tunable lens with a conventional microscope with modified illumination, for motion-free, high spatial resolution real-time all-in-focus imaging over thick volumes. EDOF imaging is obtained with spatial resolution up to near the incoherent diffraction limit (similar to 388 nm, 20x / 0.8NA) and temporal resolution similar to 30 fps. Moreover, it is demonstrated that the coupled phase and absorption components of the complex refractive index in optical diffraction tomography microscopy can be solved by focus scanning. The Richardson-Lucy deconvolution with total variation regularization is adopted for deblurring and suppressing noise under low light efficiency high-speed exposure. To demonstrate the capabilities of the proposed method, experiments are conducted using fixed transgenic zebrafish larvae, Drosophila larvae and dynamic Caenorhabditis elegans under both transmissive and fluorescent imaging modalities, revealing the proposed approach is prospective to be adopted by broad applications such as pharmacokinetics and tumor immunology. A new extended depth of field microscopy technique based on partially coherent annular illumination, termed AI-EDOF, has been proposed, by combining an electrically tunable lens with a conventional microscope with modified illumination, for motion-free, high spatial resolution real-time all-in-focus imaging over thick volumes. With the help of ETL, the intensity distribution of a 3D sample can be integrated along the z-axis and 'projected' onto the 2D image plane, which is related to the 'Fourier Slice Theorem'.image