One-step deconvolution for multi-angle TIRF microscopy with enhanced resolution

Total internal reflection fluorescence microscopy (TIRF microscopy) uses a rapid decay of evanescent waves to excite fluorophores within several hundred nanometers ( nm ) beneath the plasma membrane, which can effectively suppress excitation of fluorescence signals in the deep layers. From image stacks obtained with a plurality of different incident angles, a three-dimensional spatial structure of the observed sample can be reconstructed by a Multi-Angle-TIRF (MA-TIRF) algorithm that provides an axial resolution of ~50 nm . Taking into account the point spread function (PSF) of the TIRF microscopes, we further increase its lateral resolution by introducing a fast deconvolution algorithm into the reconstruction of MA-TIRF data (DMA-TIRF), which is approached in just one step of minimizing the reconstruction function. We also introduce a TV regularization term in the deconvolution algorithm to suppress artifacts induced by the excessive noise. Therefore, based on the hardware of existing MA-TIRF microscopes, the proposed DMA-TIRF algorithm has achieved lateral and axial resolutions of ~200 and ~50 nm , respectively.