Tissue Clearing and 3D Light Sheet Imaging of Neural Circuits in the Intact and Injured Spinal Cord

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Dr. Dylan McCreedy Assistant Professor,
Texas A&M University (USA)

Tissue clearing and 3D light sheet imaging have enabled detailed interrogation of neural circuits in the brain, however, corresponding studies in the spinal cord have been more limited. We have optimized aqueous- and organic solvent-based tissue clearing protocols, which has enabled high-resolution imaging of the full spinal cord using light sheet microscopy. Molecularly-defined neuronal subsets can then be readily visualized in the cleared spinal cord using multiple fluorescent labeling and reporter systems. We are currently developing high-throughput image analysis methods to generate a 3D map of neural circuits in the intact spinal cord and to characterize damage that occurs following spinal cord injury. The knowledge gained from these studies will advance our understanding of motor and sensory systems in the spinal cord and provide critical insight into repair strategies to restore function lost due to injury.

Key Learnings:

  • Comprehensive overview of how to prepare cleared mouse spinal cords for fluorescence microscopy
  • Overview of fluorescent markers and reporters used to visualize different neuronal cell subtypes within whole mouse spinal cords
  • Analysis of spinal cord injury using light sheet microscopy

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Light sheet fluorescence microscopy (LSFM) with its unique illumination principle allows fast and gentle imaging of whole living model organisms, tissues, and developing cells. The high stability of ZEISS Lightsheet 7 enables researchers to observe living samples over extended periods of time – even days – with less phototoxicity than ever before. The new light sheet microscope can also be used to image very large optically cleared specimens in toto, and with subcellular resolution. The dedicated optics, sample chambers, and sample holders of ZEISS Lightsheet 7 can be adjusted to the refractive index of the chosen clearing method to observe large samples, even whole mouse brains.