Pipeline for 4D whole-brain microscopy preprocessing and analysis.

Whole-brain microscopy allows for high-resolution imaging of intact mouse brains. It is a promising technique to relate behaviour to underlying brain activity, for example when combined with staining of immediate early genes. Multiple tools have been developed to transform images to machine-readable numbers, but these do not address the statistical requirements for robust data analysis. We present a novel pre-processing and analytical pipeline to analyze whole-brain data in 4 dimensions, from macro- to micro-scale and over time. This practical pipeline guides researchers through the appropriate cleaning, normalization and transformation steps for each analysis type. We developed analyses at different spatial resolutions, from macroscale functional networks to the single cell. We validate and showcase the advantages of the method by analysing activity changes at various time-points after foot-shock in male mice. All data can be visualized in our interactive web-portal (https://utrecht-university.shinyapps.io/brain_after_footshock/). The pipeline is available as R-package, abc4d.