Open Access

Blaž Brence, Maria Ahmed, Sajjad Khawari, Daniel Baum

• The analysis of complex imaging data often represents a significant barrier to progress in neurobiology. This is particularly true for the analysis of cells with a complex morphology, and when the imaged data is multi-dimensional. Here, we present a holistic workflow for 4D quantitative analysis of the morphogenesis of a columnar neuron in the developing Drosophila melanogaster brain. The example data shows a neuron that simultaneously contacts tens of columns in the visual neuropil, posing a significant challenge of resolving spatially heterogeneous contact dynamics across individual columns. For successful quantification, the 4D multi-channel imaging data undergoes several processing steps, including data standardization, alignment, discretization, and imaging angle correction. From the processed data, contact dynamics at the level of individual columns are extracted. To facilitate the analysis of results, we developed the Column Explorer tool, which enables the user to visualize and explore the results of the workflow. In addition, analysis of the time requirements for the execution of the workflow is provided for three datasets. The quantitative framework presented here is adaptable to other neural systems with columnar organization, thus providing a generalizable tool for studying contact dynamics during neural circuit development.