Acquiring an accurate orientation reference is a prerequisite for precisely analysing the morphological features of Golgi-stained neurons in the whole brain. However, the same reflective imaging contrast of Golgi staining for morphology and Nissl staining for cytoarchitecture leads to the failure of distinguishing soma morphology and simultaneously co-locate cytoarchitecture. Here, we developed the dual-mode micro-optical sectioning tomography (dMOST) method to simultaneously image the reflective and fluorescent signals in three dimensions. We evaluated the feasibility of real-time fluorescent counterstaining on Golgi-stained brain tissue. With our system, we acquired whole-brain data sets of physiological and pathological Golgi-stained mouse model brains with fluorescence-labelled anatomical annotation at single-neuron resolution. We also obtained the neuronal morphology of macaque monkey brain tissue using this method. The results show that real-time acquisition of the co-located cytoarchitecture reference in the same brain greatly facilitates the precise morphological analysis of Golgi-stained neurons.
Keywords: (110.0110) Imaging systems; (110.6880) Three-dimensional image acquisition; (120.5700) Reflection; (180.2520) Fluorescence microscopy.