Tissue Clearing and Light Sheet Microscopy: Imaging the Unsectioned Adult Zebra Finch Brain at Cellular Resolution.

The inherent complexity of brain tissue, with brain cells intertwining locally and projecting to distant regions, has made three-dimensional visualization of intact brains a highly desirable but challenging task in neuroscience. The natural opaqueness of tissue has traditionally limited researchers to techniques short of single cell resolution such as computer tomography or magnetic resonance imaging. By contrast, techniques with single-cell resolution required mechanical slicing into thin sections, which entails tissue distortions that severely hinder accurate reconstruction of large volumes. Recent developments in tissue clearing and light sheet microscopy have made it possible to investigate large volumes at micrometer resolution. The value of tissue clearing has been shown in a variety of tissue types and animal models. However, its potential for examining the songbird brain remains unexplored. Songbirds are an established model system for the study of vocal learning and sensorimotor control. They share with humans the capacity to adapt vocalizations based on auditory input. Song learning and production are controlled in songbirds by the song system, which forms a network of interconnected discrete brain nuclei. Here, we use the CUBIC and iDISCO+ protocols for clearing adult songbird brain tissue. Combined with light sheet imaging, we show the potential of tissue clearing for the investigation of connectivity between song nuclei, as well as for neuroanatomy and brain vasculature studies.

Expansion Light Sheet Microscopy Resolves Subcellular Structures in Large Portions of the Songbird Brain.

Expansion microscopy and light sheet imaging (ExLSM) provide a viable alternative to existing tissue clearing and large volume imaging approaches. The analysis of intact volumes of brain tissue presents a distinct challenge in neuroscience. Recent advances in tissue clearing and light sheet microscopy have re-addressed this challenge and blossomed into a plethora of protocols with diverse advantages and disadvantages. While refractive index matching achieves near perfect transparency and allows for imaging at large depths, the resolution of cleared brains is usually limited to the micrometer range. Moreover, the often long and harsh tissue clearing protocols hinder preservation of native fluorescence and antigenicity. Here we image large expanded brain volumes of zebra finch brain tissue in commercially available light sheet microscopes. Our expansion light sheet microscopy (ExLSM) approach presents a viable alternative to many clearing and imaging methods because it improves on tissue processing times, fluorophore compatibility, and image resolution.