Structural analysis of biological membranes is important for understanding cell and sub-cellular organelle function as well as their interaction with the surrounding environment. Imaging of whole cells in three dimension at high spatial resolution remains a significant challenge, particularly for thick cells. Cryo-transmission soft X-ray microscopy (cryo-TXM) has recently gained popularity to image, in 3D, intact thick cells (∼10μm) with details of sub-cellular architecture and organization in near-native state. This paper reports a new tool to segment and quantify structural changes of biological membranes in 3D from cryo-TXM images by tracking an initial 2D contour along the third axis of the microscope, through a multi-scale ridge detection followed by an active contours-based model, with a subsequent refinement along the other two axes. A quantitative metric that assesses the grayscale profiles perpendicular to the membrane surfaces is introduced and shown to be linearly related to the membrane thickness. Our methodology has been validated on synthetic phantoms using realistic microscope properties and structure dimensions, as well as on real cryo-TXM data. Results demonstrate the validity of our algorithms for cryo-TXM data analysis.

R.C. was partially funded by the Instituto de Salud Carlos III, Spain (FIS- PI11/01709), and BE-DGR 2012 BE1 00308 from AGAUR, Catalonia, Spain. C.Z. acknowledges the financial support from the Spanish Ministry of Economy and Competitiveness, through the Maria de Maeztu Programme for Centres/Units of Excellence in R&D (MDM-2015-0502). O.K. was supported by the Seventh Framework Program of the European Commission, grant agreement 278486: DEVELAGE. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N283570), the Spanish Ministry of Economy and Competitiveness (grant TIN2014-52923-R) and FEDER.