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

Abstract

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.

Fast retrograde access to projection neuron circuits underlying vocal learning in songbirds

“…To date, in vivo imaging of dendritic spine plasticity in songbirds has been achieved exclusively via lentivirus-mediated fluorescence labeling 12,26 , a tool that lacks retrograde transduction capabilities. Furthermore, the strong eGFP expression we demonstrate is highly beneficial for tissue processing techniques that require a strong initial fluorophore expression, including large-volume expansion (lattice) light-sheet microscopy (ExLSM 27 , ExLLSM 28 ) and whole-brain tissue clearing 29 .…”

Advances in Ex Situ Tissue Optical Clearing

“…As expected, optical clearing is limited neither to brain, nor peripheral organs of mammals and as such was already successfully applied to morphological studies of other model vertebrate organisms spheroids, plant tissues, as well as insects and crustaceans and even implant‐tissue interface . Interestingly, amorphous samples such as blood clot or sputum from cystic fibrosis patients can also be cleared and further visualized in situ to reveal spatial distribution of bacteria with associated proteins and nucleic acids and therefore to decipher their otherwise inaccessible microenvironment.…”

FalseColor-Python: A rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology

“…For example, techniques such as confocal microscopy [1,2], multiphoton microscopy [3][4][5][6], microscopy with UV surface excitation (MUSE) [7][8][9], and structured illumination microscopy (SIM) [10,11] have been explored as slide-free alternatives to frozen-section histology for rapid interoperative guidance. Additionally, light-sheet microscopy technologies, such as open-top light-sheet (OTLS) microscopy [12][13][14], when used in conjunction with tissue-clearing techniques [15], have been explored for slide-free nondestructive 3D pathology. These techniques generally rely on fluorescence collected with a sensitive monochrome detector.…”