Automated neuronal reconstruction with super-multicolour fluorescence imaging

Fluorescence imaging is widely used for the mesoscopic mapping of neuronal connectivity. However, neurite reconstruction is challenging, especially when neurons are densely labelled. Here we report a strategy for the fully automated reconstruction of densely labelled neuronal circuits. Firstly, we established stochastic “super-multicolour” labelling with up to seven different fluorescent proteins using the Tetbow method. With this method, each neuron was labelled with a unique combination of fluorescent proteins, which were then imaged and separated by linear unmixing. We also established an automated neurite reconstruction pipeline based on the quantitative analysis of multiple dyes (QDyeFinder). To classify colour combinations, we used a newly developed unsupervised clustering algorithm, dCrawler, in which data points in multi-dimensional space were clustered based on a given threshold distance. Our new strategy allows for the reconstruction of neurites for up to hundreds of neurons at a millimetre scale without manual tracing. ### Competing Interest Statement TI, MNL, and SF have filed a patent application for QDyeFinder.