Alternative Methods to Enhance the Axial Resolution of Total Internal Reflection Fluorescence–Structured Illumination Microscopy

Total internal reflection fluorescence–structured illumination microscopy (TIRF-SIM) can enhance the lateral resolution of fluorescence microscopy to twice the diffraction limit, enabling subtler observations of activity in subcellular life. However, the lack of an axial resolution makes it difficult to resolve three-dimensional (3D) subcellular structures. In this paper, we present an alternative TIRF-SIM axial resolution enhancement method by exploiting quantitative information regarding the distance between fluorophores and the surface within the evanescent field. Combining the lateral super-resolution information of TIRF-SIM with reconstructed axial information, a 3D super-resolution image with a 25 nm axial resolution is achieved without attaching special optical components or high-power lasers. The reconstruction results of cell samples demonstrate that the axial resolution enhancement method for TIRF-SIM can effectively resolve the axial depth of densely structured regions.