Abstract
Brain circuits are highly interconnected three-dimensional structures fabricated from components ranging vastly in size; from cell bodies to individual synapses. While neuronal activity can be visualized with advanced light microscopy (LM) techniques, the resolution of electron microscopy (EM) is critical for identifying synaptic connections between neurons. Here, we combine these two techniques, affording the advantage of each and allowing for measurements to be made of the same neural features across imaging platforms. We established an EM-label-free workflow utilizing inherent structural features to correlate in vivo two-photon LM and volumetric scanning EM (SEM) in the ferret visual cortex. By optimizing the volume SEM sample preparation protocol, imaging with the OnPoint detector, and utilizing the focal charge compensation device during serial block-face imaging, we achieved sufficient resolution and signal-to-noise ratio to analyze synaptic ultrastructure for hundreds of synapses within sample volumes. Our novel workflow provides a reliable method for quantitatively characterizing synaptic ultrastructure in functionally imaged neurons, providing new insights into neuronal circuit organization.
Highlights
To understand how the brain processes and encodes information, neuronal networks must be investigated both functionally and anatomically
Overview of Workflow to Correlate In Vivo 2-Photon Imaging and SBF-scanning EM (SEM)
We trimmed each slice to a smaller square, placing the target cell in the center using blood vessels as fiducial markers (Figs. 1d, 3), performed heavy metal staining with our optimized protocol (Figs. 1e, 2 and Table 1)
Summary
To understand how the brain processes and encodes information, neuronal networks must be investigated both functionally and anatomically. In light microscopy (LM), the development of activity-dependent fluorophores, such as GCaMP (Nakai et al, 2001), and improvements to depth and resolution of live imaging techniques enable quantification of neuronal function at the level of individual dendritic spines, a major site of cell-to-cell communication (Ji et al, 2016; Lu et al, 2017; Scholl et al, 2017; Luo et al, 2018; Moyer & Zuo, 2018).
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