Serial two-photon tomography for automated ex vivo mouse brain imaging

Here we describe an automated method, which we call serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in 3D, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders. Since the pioneering work of Ramón y Cajal, advances in light microscopy have been central to many discoveries in neuroanatomy. The recent focus on systematic generation of whole-brain datasets, for example the Allen Mouse Brain Atlas for gene expression1 and the ongoing Mouse Brain Architecture Project for mesoscopic connectivity2, has created a pressing need for the development of new instrumentation for high-throughput whole-brain imaging. Here we describe a microscopy method, which we call serial two-photon (STP; pronounced “step”) tomography, that achieves automated high-throughput imaging of fluorescently labeled mouse brains. This method uses whole-mount two-photon microscopy3, 4 and thus generates datasets of well aligned, high-resolution serial optical sections. We tested the versatility of STP tomography by imaging four mouse brains with cell type-specific fluorescent protein expression and systematically mapping input and output connections of mouse somatosensory cortex. These experiments show that STP tomography is a robust imaging method that can transform the emerging field of systematic whole-brain anatomy, until now limited to dedicated atlasing initiatives1, 2, into a routine methodology applicable, for example, to the study of mouse models of human brain disorders in standard laboratory settings