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Serial Multiphoton Tomography and Analysis of Volumetric Images of the Mouse Brain

  • Denise M. O. RamirezEmail author
  • Apoorva D. Ajay
  • Mark P. Goldberg
  • Julian P. Meeks
Protocol
Part of the Neuromethods book series (NM, volume 148)

Abstract

Mapping the structural and synaptic organization of the central nervous system is fundamental to a principled understanding of neural circuit development and function and an important goal of modern neuroscience. A plethora of new imaging technologies and computational advances have made whole-brain mapping studies more widely accessible. One such volumetric imaging method is known as serial two-photon tomography (STPT). STPT is an automated block-face imaging method in which a brain or other whole-organ specimen is repetitively imaged using multiphoton illumination and physically sectioned using an integrated vibratome. The resultant tile images are stitched in two dimensions to form mosaic whole-section images, and the mosaic images need only be stacked in three dimensions to generate a whole-brain volumetric image. Automated image analysis pipelines may then be employed to mine quantitative information at the whole-brain scale across large cohorts of experimental animals. Here, we describe our methods optimized in the University of Texas Southwestern Whole Brain Microscopy Facility for STPT using the TissueCyte1000 platform and a custom pipeline for whole-brain image analysis including registration into the Allen Institute Common Coordinate Framework version 3.0 (CCF 3.0). Included is a description of the inclusion of supervised machine learning using a voxel-wise random forest model for classification of features of interest, including cell bodies and subcellular structures. The rapidly advancing pace of STPT and other complementary methods for whole-brain mapping and systematic analysis has the potential to generate transformative insights into brain circuitry in both health and disease.

Keywords

TissueCyte Volumetric imaging Automated image analysis Block-face imaging Serial two-photon tomography Connectomics 

Notes

Acknowledgements

The University of Texas Southwestern Whole Brain Microscopy Facility is funded by the Texas Institute of Brain Injury and Repair (TIBIR) and receives additional support from the University of Texas Southwestern Department of Neurology and Neurotherapeutics and the University of Texas Southwestern Center for Alzheimer’s and Neurodegenerative Disease (CAND). We would like to thank Drs. Amy Bernard, Anh Ho, Lydia Ng, and Hongkui Zeng of the Allen Institute for Brain Science for helpful discussions and training in TissueCyte operations and data analysis. We also thank Drs. Timothy Ragan, Phil Knodle, and Adam Bleckert of TissueVision, Inc. for assistance in optimizing Autostitcher performance.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Denise M. O. Ramirez
    • 1
    • 2
    Email author
  • Apoorva D. Ajay
    • 1
    • 2
  • Mark P. Goldberg
    • 1
    • 2
  • Julian P. Meeks
    • 1
    • 2
    • 3
  1. 1.Department of Neurology and NeurotherapeuticsUniversity of Texas Southwestern Medical CenterDallasUSA
  2. 2.Peter O’Donnell, Jr. Brain InstituteUniversity of Texas Southwestern Medical CenterDallasUSA
  3. 3.Department of NeuroscienceUniversity of Texas Southwestern Medical CenterDallasUSA

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