FIGURE 2.

Long-term transcranial TPLSM imaging of fine neuronal structures. (A) CCD camera view of brain vasculature under the thinned skull. The cortical vasculature can clearly be seen through the thinned skull. The vasculature pattern remains stable over months to years and can be used as a landmark to relocate the imaged region at subsequent time points. Arrow indicates the region in which subsequent TPLSM images were obtained. (B) Two-dimensional (2D) projection of a three-dimensional (3D) stack of dendritic branches and axons in the primary visual cortex (60×, ~0.39 µm per pixel). The stack was 50-µm deep (2-µm step size). The boxed region was then imaged at higher magnification. (C) High-power 2D projection of a 3D stack (60×, ~0.13 µm per pixel, 10-µm reconstructed, 0.70-µm step size) reveals clear neuronal structures, including axonal varicosities, dendritic shafts, and dendritic protrusions. (D,E) Axonal and dendritic branches from two animals imaged 3 d apart show the same spines and boutons at the same locations. (F,G) Dendritic branches imaged over 19 mo apart. The arrows indicate spines that are eliminated in the second view. The arrowheads indicate spines that are formed in the second view. Note that most spines in F persist in G. Two-dimensional projections of 3D image stacks containing dendritic segments of interest were used for D–G.