Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution

Abstract

Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain. However, optical microscopy offer insufficient resolution to reveal subcellular details and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.

Light microscopy based approach for mapping connectivity with molecular specificity

“…Measuring synapses using conventional light microscopy (LM) techniques is difficult as the distance between synaptic structures and neuronal boundaries can be smaller than the diffraction limit 6 . Recently, expansion microscopy has been shown to be a viable strategy for resolving synaptic structures and assigning them to neurons 13,27,28 . Consequently, we combined Brainbow with miriEx to measure synaptic structures located at the junctions between different neurons in an effort to define connectivity using LM.…”

Light microscopy of proteins in their ultrastructural context

“…Our sample preparation is completed in a few days; subsequent imaging takes less than a minute (2D image) to tens of minutes (3D data set; Supplementary Video 2 ) per cell on a conventional light microscope. We anticipate that light-sheet microscopes or other instruments optimized for ExM [15] will accelerate future pan-ExM data acquisition substantially.…”

TeraVR Empowers Precise Reconstruction of Complete 3-D Neuronal Morphology in the Whole Brain

“…With the accurate pinpointing capability in TeraVR ( Supplementary Fig. 2), in real-time a user can precisely and efficiently 8 load the data of a desired high-resolution ROI to see detailed 3-D morphological structures ( Supplementary Fig. 2c).…”

“…While immersive VR visualization of biological surface objects and sometimes also imaging data were shown in applications such as biological education and data analyses ( Supplementary Table 2), there is little existing work on developing open-source VR software packages for very complicated and teravoxel-scale imaging datasets such as the whole brain 20 imagery as we have introduced here. We expect that TeraVR can also be used to analyze other massive-scale datasets especially those produced using fast or highresolution microscopy methods, such as the light-sheet microscopy (Keller, et al, 2008;Ahrens, et al, 2013;Silvestri, et al, 2013), expansion-microscopy (Chen, et al, 2015), and recent nanoscale lattice microscopy (Gao, et al, 2019).…”

“…LMreconstruction makes it possible to trace both long projections and the terminal arborization of a brain cell. Recent extension of this approach based on expansion microscopy can help visualize neurons at nanometer-resolution using LM approaches (Gao, 2019).…”