Abstract
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.