Label-Free Visualization of Ciliary Rootlets in Mouse Brain

Neuronal primary cilia play important roles in brain development, sensory perception, and neurogenesis. Rootletin, a fibrous protein composed of coiled-coil motifs, is a key structural component of ciliary rootlets and is essential for understanding ciliary functions. However, the precise mechanisms by which Rootletin influences ciliary dynamics and impacts neuronal function remain largely unknown, primarily due to the challenges associated with visualizing these structures. In this study, we present a label-free, rapid, and highly sensitive method for visualizing Rootletin molecules in brain tissue. This platform integrates a second harmonic generation (SHG) microscope with background reduction by a one-step chemical pretreatment. Additionally, we employed coherent anti-Stokes Raman scattering imaging to simultaneously determine the cellular regions and intracellular locations of SHG signals. Applying this multimodal multiphoton imaging approach to the mouse hippocampus revealed that neuronal ciliary rootlets exhibited highly organized and specific intracellular distributions. Moreover, measurements of cultured neurons revealed that ciliary rootlets were detected even in immature neurons. These findings highlight the utility of our label-free imaging platform in developmental and neuroscience research, providing a powerful tool for characterizing ciliary dynamics and neuronal function.