Combination of nanometer-scale 3D structural analysis with optical characterization of the same material is a challenging task. Its results may be important for nanophotonics, materials science, and quality control. We have developed a new technique for complementary high-resolution structural and optical characterization followed by optical spectroscopic and microscopic measurements accompanied by reconstruction of the 3D structure in the same area of the sample. The 3D structure is reconstructed by combination of ultramicrotomic and SPM techniques allowing the study of the 3D distribution of implanted nanoparticles and their effect on the matrix structure. The combination of scanning probe nanotomography (SPN) and optical microspectroscopy makes it possible to direct estimate how the 3D structural characteristics of materials affect their macroscopic optical properties. The technique developed has been applied to the engineering of materials made from cholesteric liquid crystals and fluorescent quantum dots (QDs). These materials permit photochemical patterning and image recording through the changes in the dissymmetry factor of circular polarization of QD emission. The differences in the polarisation images and morphological characteristics of the liquid crystal matrix have proved to be correlated with the arrangement of the areas of homogeneous distribution and nonhomogeneous clustering of QDs. The reconstruction of the 3D structure of the liquid crystal matrix in the areas of homogeneous QD distribution has shown that QDs embedded into cholesteric liquid crystal matrices do not perturb their periodic planar texture. The combined optical/SPM/ultramicrotome technique will be indispensable for evaluating the effects of inorganic nanoparticles on the organisation of organic and liquid crystal matrices, biomedical materials, cells, and tissues.