Ultrabright chemical labeling enables rapid neural connectivity profiling in large tissue samples

Comprehensive mapping of neuronal connections across entire nervous systems remains a fundamental challenge in neuroscience. Here, we introduce labeling individual neurons with chemical dyes and controllable sparseness (LINCS), a technology that achieves rapid, ultrabright, and photostable labeling of specific cell types throughout the entire mouse brain and body. LINCS utilizes an engineered, solubility-enhanced biotin ligase for in vivo biotinylation, followed by rapid whole-mount staining with a high-affinity monovalent streptavidin. When integrated with tissue clearing and light-sheet microscopy, this system creates an efficient pipeline for profiling long-range neuronal projections across both the central and peripheral nervous systems. Furthermore, we developed an adeno-associated virus (AAV) strategy employing Cas9-mediated Cre knockout to achieve stable sparse labeling, permitting the precise morphological reconstruction of individual neurons at scale. The LINCS toolkit substantially lowers the barrier to large-scale connectivity mapping and will accelerate the anatomical and functional dissection of mammalian neural circuits.