Rapid, label-free identification of cerebellar structures using multiphoton microscopy.

The cerebellum is the prominent laminar structure of the mammalian brain that has been implicated in various psychiatric and neurological diseases. Although clinical brain imaging techniques have provided precise anatomic images of cerebellar structures, a definitive diagnosis still requires adequate resolution to identify individual layers in cerebellar cortex, the extent of tumor, even requires the histological tissue examination during surgical procedures. In this study, multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), was perform on the rat cerebellar structures and pathology with the combination of image analysis methods. Results show that MPM can reveal the cerebellar vermis, hemispheres, medulla, and ventricle, as well as axon bundles, Purkinje cells, capillaries, and the pia mater of the cerebellum. Together with custom-developed image processing algorithms, MPM could further differentiate between the gray and white matter, as well as evaluate the Purkinje cell layer, identify the cerebellar tumor boundary, and distinguish between the tumor core and peritumor regions. Our results establish a direct visualization and rapid assessment approach for the cerebellar structures, as well as suggest the feasibility of in vivo multiphoton microendoscopes and fiberscopes as clinical tools for neuropathological diagnoses.