A software tool for tomographic axial superresolution in STED microscopy

A method for generating three-dimensional tomograms from multiple three-dimensional axial projections in STimulated Emission Depletion (STED) superresolution microscopy is introduced. Our STED< method, based on the use of a micromirror placed on top of a standard microscopic sample, is used to record a three-dimensional projection at an oblique angle in relation to the main optical axis. Combining the STED< projection with the regular STED image into a single view by tomographic reconstruction, is shown to result in a tomogram with three-to-four-fold improved apparent axial resolution. Registration of the different projections is based on the use of a mutual-information histogram similarity metric. Fusion of the projections into a single view is based on Richardson-Lucy iterative deconvolution algorithm, modified to work with multiple projections. Our tomographic reconstruction method is demonstrated to work with real biological STED superresolution images, including a data set with a limited signal-to-noise ratio (SNR); the reconstruction software (SuperTomo) and its source code will be released under BSD open-source license. Lay Description STimulated Emission Depletion (STED) microscopy provides intrinsic subdiffraction resolution in far-field fluorescence microscopy. However, the standard lateral donut' STED depletion beam only improves the resolution in the lateral direction, while the axial dimension has the same resolution as a regular confocal microscope. In a typical STED system lateral resolution, the minimum distance between two adjacent image features can be as low as few tens of nanometres, but the axial resolution is limited to 500600 nm - a mismatch of more than an order of magnitude. We propose a new method to deal with this problem, on such axially challenged STED systems, which does not require any modifications of the microscope optics. It is based on tomographic reconstruction of multiple three-dimensional axial projections. Our own STED< method, based on the use of a micromirror placed on top of a standard microscopic sample, is used to record a three-dimensional (3D) projection at an oblique angle in relation to the main optical axis. Combining the STED< projection with the regular STED image into a single view by tomographic reconstruction, is shown to result in a tomogram with significantly improved apparent axial resolution. Our automatic tomographic reconstruction method is demonstrated to work with real biological STED superresolution images, including a data set with a very limited SNR; the reconstruction software (SuperTomo) and its source code will be released under BSD open-source license.