The paper reports confocal laser scanning microscopy and scanning electron microscopy complex study of polyamide-6 scaffolds for tissue engineering. Various approaches of 3D reconstruction and analysis of porous space and fibrous structure are presented. The effective porosity and cell penetration into the scaffold are estimated, assuming the spherical shape of cells. These results are compared with the data, obtained by bubble point method. The average diameter of the fibers was estimated using both skeletonization and manual approach, and it was within the range of 3.4–4.1 μm. Cell penetration modeling revealed that the effective volume of the porous space depends on the size of the penetrating sphere, decreasing from nearly 90% for 6-μm sphere to 0.2% for 24-μm one. The influence of experimental conditions, namely 405, 473, 559, and 635-nm laser wavelength, different step sizes during laser scanning and media (distilled water, glycerol 70%, glycerol 90%, glycerol 100%, and cultural fluid RPMI-1648), on the results obtained by confocal laser scanning microscopy is considered. It was found strong dependence of laser wavelength on image quality. © 2017, Springer Science+Business Media, LLC, part of Springer Nature.