Quantification of the structure and composition of biomaterials using micro-CT requires image segmentation due to the low contrast and overlapping radioopacity of biological materials. The amount of bias introduced by segmentation procedures is generally unknown. We aim to develop software that generates three-dimensional models of fibrous and porous structures with known volumes, surfaces, lengths, and object counts in fibrous materials and to provide a software tool that calibrates quantitative micro-CT assessments. Virtual image stacks were generated using the newly developed software TeIGen, enabling the simulation of micro-CT scans of unconnected tubes, connected tubes, and porosities. A realistic noise generator was incorporated. Forty image stacks were evaluated using micro-CT, and the error between the true known and estimated data was quantified. Starting with geometric primitives, the error of the numerical estimation of surfaces and volumes was eliminated, thereby enabling the quantification of volumes and surfaces of colliding objects. Analysis of the sensitivity of the thresholding upon parameters of generated testing image sets revealed the effects of decreasing resolution and increasing noise on the accuracy of the micro-CT quantification. The size of the error increased with decreasing resolution when the voxel size exceeded 1/10 of the typical object size, which simulated the effect of the smallest details that could still be reliably quantified. Open-source software for calibrating quantitative micro-CT assessments by producing and saving virtually generated image data sets with known morphometric data was made freely available to researchers involved in morphometry of three-dimensional fibrillar and porous structures in micro-CT scans.

Biomedical Center Faculty of Medicine in Pilsen Charles University Pilsen 323 00 Czech Republic

Department of Composites and Carbon Materials Institute of Rock Structure and Mechanics Academy of Sciences of the Czech Republic Prague 8 Czech Republic

Department of Histology and Embryology Faculty of Medicine in Pilsen Charles University Prague Pilsen 301 66 Czech Republic

European Centre of Excellence NTIS Faculty of Applied Sciences University of West Bohemia Univerzitní 22 Pilsen 306 14 Czech Republic

Faculty of Textile Engineering Technical University of Liberec Liberec 1 461 17 Czech Republic

Institute of Dental Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Prague 2 128 01 Czech Republic

Institute of Pathological Physiology 1st Faculty of Medicine Charles University U nemocnice 5 128 53 Prague Czech Republic

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