A software tool for tomographic axial superresolution in STED microscopy

S Koho; T Deguchi; P E Hänninen

doi:10.1111/jmi.12287

A software tool for tomographic axial superresolution in STED microscopy

J Microsc. 2015 Nov;260(2):208-18. doi: 10.1111/jmi.12287. Epub 2015 Aug 10.

Authors

S Koho¹, T Deguchi¹, P E Hänninen¹

Affiliation

¹ Department of Cell Biology and Anatomy, Laboratory of Biophysics, Institute of Biomedicine and Medicity Research Laboratories, University of Turku, Tykistökatu 6A, Turku, Finland.

PMID: 26258639
DOI: 10.1111/jmi.12287

Abstract

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.

Keywords: Axial tomography; STED; image fusion; image processing; image registration; open source software; reconstruction algorithms; superresolution microscopy.