Development of a fluorescence contrasting immunostaining technique for visualizing 3D astrocytic ultramorphology

Changes in astrocytic ultramorphology may underlie the development of neurodegenerative processes in their early stages. However, the mechanisms of its change are still poorly understood, since the size of the peripheral astrocytic processes forming the basis of the astrocytic synaptic coating are beyond the resolution of most optical microscopy (OM) methods. In turn, the disadvantage of promising methods of electron and scanning probe microscopy (EM and SPM) for such studies is the inability to determine the target area of the study due to the simultaneous use of fluorescence microscopy of immunocolored cells and the possibility of full-fledged 3D analysis of samples. In this paper, we consider the concept of solving the above problem by using an instrumental approach that combines the methods of SPM and OM together with ultramicrotomy as a method of restoring the 3D structure of the sample within a single hardware complex. To implement the proposed combined technique (optical-probe nanotomography, OPNT), the first stage of creating specialized fluorescent-contrasting labels based on conjugates of fluorescent semiconductor nanocrystals and single-domain antibodies has been developed in this work. This type of label will provide both immuno-staining of the “area of interest" for the restoration of 3D astrocytic ultramorphology, and contrast of astrocytes by the SPM method.