Semi-automatic Geometrical Reconstruction and Analysis of Filopodia Dynamics in 4D Two-Photon Microscopy Images

Filopodia are thin and dynamic membrane protrusions that play a crucial role in cell migration, axon guidance, and other processes where cells explore and interact with their surroundings. Historically, filopodial dynamics have been studied in great detail in 2D in cultured cells, and more recently in 3D culture as well as living brains. However, there is a lack of efficient tools to trace and track filopodia in 4D. To address this issue, we have developed a semi-automatic workflow for tracing filopodia in 3D images and tracking the traced filopodia over time.

The workflow was developed based on high-resolution data of photoreceptor axon terminals in the in vivo context of normal Drosophila brain development, but devised to be applicable to filopodia in any system, including at different temporal and spatial scales. The workflow was realized within the Amira software system and consists of two main parts, dataset pre-processing, and geometrical filopodia reconstruction, where each of the two parts comprises multiple steps. In this paper we provide an extensive workflow description and demonstrate its versatility for different axo-dendritic morphologies. Finally, we provide an analysis of the time requirements for use input and data processing.

To facilitate simple application within Amira or other frameworks, we share the source code, which is available at https://github.com/zibamira/filopodia-tool .