Uncovering brain tissue architecture across scales with super-resolution light microscopy

Abstract

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the system's molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.

Expansion Microscopy: Super-Resolution Imaging with Hydrogels

“…Recently, Michalska et al developed CATS ( C omprehensive A nalysis of T issue across S cales) to visualize glycans of the extracellular space and utilized it for fluorescence-based tracing of brain tissue architecture across scales . The CATS protocol accesses glycans by flooding the extracellular space of a living specimen, e.g., an acute brain slice, with WGA ( W heat- G erm A gglutinin), which binds to the extracellular glycan components N-acetyl- d -glucosamine and sialic acid .…”

“…Recently, Michalska et al developed CATS ( C omprehensive A nalysis of T issue across S cales) to visualize glycans of the extracellular space and utilized it for fluorescence-based tracing of brain tissue architecture across scales . The CATS protocol accesses glycans by flooding the extracellular space of a living specimen, e.g., an acute brain slice, with WGA ( W heat- G erm A gglutinin), which binds to the extracellular glycan components N-acetyl- d -glucosamine and sialic acid . As WGA is poorly retained via standard protein-retention strategies for expansion microscopy, , due to a relative lack of lysines and therefore primary amines that could react to the anchoring agents, CATS employs WGA coupled to biotin.…”

“…Less than eight years ago, at the time of writing, expansion microscopy was introduced as a proof of concept on proteins in cell culture and brain slices, attaining a limited resolution of ∼70 nm. 1 Today, expansion microscopy is applicable to any biomolecule 24,27,28,[46][47][48][49]95 and most model organisms, 22,26,[35][36][37][38][39][40]119,128 and it attains 25 nm resolution and The techniques are listed in approximate order of publication. Note that this list is not exhaustive, it merely contains the contributions most pertinent to this review.…”

“…The biotin is then detected via streptavidin, which is rich in lysines�which are conjugated with fluorophores (for detection) and acrylamide (for gel retention) before being applied to the sample. As CATS potentially facilitates fluorescence-based connectomics studies using expansion microscopy, 95 it is likely to drive renewed interest in labeling glycans for fluorescence microscopy.…”

“…The rapid development of expansion microscopy techniques highlighted in this reviewwith myriad gel chemistries ,,− ,,,, and adaptations to access different biomolecules ,,,,− ,, and biological specimens ,− ,, being introduced on an almost weekly basisshould not make us forget that this is still a young technology. Significant challenges remain, in many areas.…”