Presynaptic actin nanostructures: A reproducibility case study

In an effort to assess the reproducibility of bioimage analyses in current publications, we took part in a Global BioImage Analysts' Society (GloBIAS) initiative to try and reproduce results from published articles. We attempted to reproduce core findings from Bingham et al. (2023), who investigate the actin organisation in presynaptic structures by using diffraction-limited and super-resolution microscopy. While the original paper unveiled novel biological insight, it lacked sufficient detail in the bioimage analysis methodology, limiting the depth of reproducibility we could achieve. Through frequent contacts with the corresponding author, we were able to replicate qualitative aspects of the analysis of actin nanostructures in bead-induced presynapses. We performed image reconstruction from super-resolution microscopy data, automatic image registration and visual inspection, followed by manual annotation of structures of interest in approximately 35 images. From this exercise, we provide concrete evidence that key practises such as sharing of example datasets, depositing manual annotations and documenting manual decision criteria and consensus procedures are essential for making bioimage analysis workflows reproducible. Our experience further highlights that transparent data sharing, adherence to bioimage analysis standards, and close collaboration between experimentalist and bioimage analysis specialists are critical to ensure the reproducibility of today's complex biological imaging studies.