Bioinformatics Spectral imaging toolbox

Background: Spectral imaging with polarity-sensitive fluorescent probes enables the quantification of cell and model membrane physical properties, including local hydration, fluidity, and lateral lipid packing, usually characterized by the generalized polarization (GP) parameter. With the development of commercial microscopes equipped with spectral detectors, spectral imaging has become a convenient and powerful technique for measuring GP and other membrane properties. The existing tools for spectral image processing, however, are insufficient for processing the large data sets afforded by this technological advancement, and are unsuitable for processing images acquired with rapidly internal-ized fluorescent probes. Results: Here we present a MATLAB spectral imaging toolbox with the aim of overcoming these limitations. In addition to common operations, such as the calculation of distributions of GP values, generation of pseudo-colored GP maps, and spectral analysis, a key highlight of this tool is reliable membrane segmentation for probes that are rapidly internal-ized. Furthermore, handling for hyperstacks, 3D reconstruction and batch processing facilitates analysis of data sets generated by time series, z-stack, and area scan microscope operations. Finally, the object size distribution is deter-mined, which can provide insight into the mechanisms underlying changes in membrane properties and is desirable for e.g. studies involving model membranes and surfactant coated particles. Analysis is demonstrated for cell membranes, cell-derived vesicles, model membranes, and microbubbles with environmentally-sensitive probes Laurdan, carboxyl-modified Laurdan (C-Laurdan), Di4-ANEPPDHQ, and Di-4-AN(F)EPPTEA (FE), for quantification of the local lateral density of lipids or lipid packing. Conclusions: The Spectral Imaging Toolbox is a powerful tool for the segmentation and processing of large spectral imaging datasets with a reliable method for membranes segmentation and no ability in programming required. For re-viewers the software is available at: https://ora.ox. ac.uk/objects/uuid:4375842f-3598-418d-8aa39b31f5023401. It will be further available on the MATLAB file exchange following acceptance for publication. Corresponding Author: Miles Aron University of Oxford UNITED KINGDOM Corresponding Author Secondary Information: Corresponding Author's Institution: University of Oxford Corresponding Author's Secondary Institution: Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation First Author: Miles Aron First Author Secondary Information: Order of Authors: Miles Aron Richard Browning Dario Carugo Erdinc Sezgin Jorge Bernardino de la Serna Christian Eggeling Eleanor Stride Order of Authors Secondary Information: Author Comments: For reviewers the software is available at: https://ora.ox. ac.uk/objects/uuid:4375842f3598-418d-8aa3-9b31f5023401. It will be further available on the MATLAB file exchange following acceptance for publication. Response to Reviewers: The authors' response letter has been included as a supplementary file Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation