Light-Sheet Fluorescence Microscopy (LSFM) is an excellent tool to study neuronal populations in large samples such as the whole mouse brain, thanks to its inherent three-dimensional nature and its high resolution down to the single cell. In this work we report on our latest technological developments in both hardware and software and their applications in several projects aiming at reconstructing the neuronal organization in both the human cortex and in whole mouse brain.Our latest LSFM setup is capable of simultaneous excitation and acquisition at four wavelengths, thus enabling fast reconstruction of the different neuronal populations in human brain tissue marked with specific immunofluorescence labels. On the whole mouse brain and using a different setup, our imaging pipeline enables cell counting across the whole organ. We have successfully demonstrated the imaging and analysis pipeline for cfos-based activation mapping in the formation of fear memory in the mouse and, in a new project, we will further apply the pipeline to map the active neuronal population during chronic exposure to, and withdrawal from, cigarette smoke in mice.On the software side, we describe our dedicated processing pipeline that was specifically designed to be able to handle the large datasets produced with light-sheet microscopy. In particular the latest additions concern the adoption of new file formats such as ome-zarr, the adoption of the Brain Imaging Data Structure (BIDS) specification (which we contributed extending for microscopy), and interactive visualization and cloud-friendly tools such as Google’s Neuroglancer.