Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Abstract

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

Advanced CLARITY for rapid and high-resolution imaging of intact tissues

“…A light sheet microscope consists of a standard wide field detection optical arm, which includes the detection objective, the tube lens and a camera, and the orthogonally-arranged independent illumination arm consisting of a low NA objective, tube lens and either a cylindrical lens to generate a static light sheet or galvanometer-scanners/f-theta lens for creating dynamic light sheets with a Gaussian or Bessel beams 8,18,24–26,29 . Conventional light sheet microscopy suffers from image quality degradation due to out-of-focus scattered light; several methods have been developed to help reject out-of-focus light in light-sheet (at some cost in imaging speed, increased photo-bleaching and instrumentation complexity), such as structured illumination 27,28 .…”

A Review of Intrinsic Optical Imaging Serial Blockface Histology (ICI-SBH) for Whole Rodent Brain Imaging

“…This allows the light to traverse the whole tissue section without much degradation and thus enable imaging, for example, of intact whole mouse brains. Some microscope configurations that exploit tissue clearing are optical projection tomography [122][123][124], light-sheet microscopy [125][126][127][128][129][130], clearing assisted scattering tomography [111], optical frequency domain imaging [111], and expansion super-resolution microscopy [131]. Despite the opportunity offered by tissue clearing to perform an optical imaging of complete whole brains, this process presents a few drawbacks such as a long preparation time, tissue shrinkage, protein and lipid loss, and the usage of highly toxic chemical reagents [132,133].…”

Data integration through brain atlasing: Human Brain Project tools and strategies

“…Fig. 3 shows a dataset acquired with high-resolution light sheet microscopy following tissue clearing [33]. The resulting 3D dataset was initially globally registered to the Allen Mouse Brain Atlas, before a local registration procedure centered on a region of the cerebral cortex to increase the precision of the positioning.…”