Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

The examination of tissue histology by light microscopy is a fundamental tool for investigating the structure and function of organs under normal and disease states. Many current techniques for tissue sectioning, imaging and analysis are time-consuming, and they present major limitations for 3D tissue reconstruction. The introduction of methods to achieve the optical clearing and subsequent light-sheet laser scanning of entire transparent organs without sectioning represents a major advance in the field. We recently developed a highly reproducible and versatile clearing procedure called 3D imaging of solvent-cleared organs, or 3DISCO, which is applicable to diverse tissues including brain, spinal cord, immune organs and tumors. Here we describe a detailed protocol for performing 3DISCO and present its application to various microscopy techniques, including example results from various mouse tissues. The tissue clearing takes as little as 3 h, and imaging can be completed in ∼45 min. 3DISCO is a powerful technique that offers 3D histological views of tissues in a fraction of the time and labor required to complete standard histology studies.