Abstract
.The goal of understanding the architecture of neural circuits at the synapse level with a brain-wide perspective has powered the interest in high-speed and large field-of-view volumetric imaging at subcellular resolution. Here, we developed a method combining tissue expansion and light-sheet fluorescence microscopy to allow extended volumetric super resolution high-speed imaging of large mouse brain samples. We demonstrate the capabilities of this method by performing two color fast volumetric super resolution imaging of mouse CA1 and dentate gyrus molecular-, granule cell-, and polymorphic layers. Our method enables an exact evaluation of granule cell and neurite morphology within the context of large cell ensembles spanning several orders of magnitude in resolution. We found that imaging a brain region of in super resolution using light-sheet fluorescence expansion microscopy is about 17-fold faster than imaging the same region by a current state-of-the-art high-resolution confocal laser scanning microscope.
Highlights
One of the prime goals in today’s neuroscience is the volumetric architectural mapping of neural circuits spanning several magnitudes of resolution, i.e., imaging large fields of view up to several millimeters in super resolution, preferably using a single, straightforward light microscopic process
We focused on a super resolution analysis of large GFP-labeled granule cells ensembles in mouse dorsal DG
Coronal DG sections were prepared for tissue expansion and light-sheet fluorescence microscopy (LSFM) analysis from a PROX1-cre mouse injected with rAAV-DIO-EGFP-WPRE to achieve selective expression of EGFP in DG granule cells
Summary
One of the prime goals in today’s neuroscience is the volumetric architectural mapping of neural circuits spanning several magnitudes of resolution, i.e., imaging large fields of view up to several millimeters in super resolution, preferably using a single, straightforward light microscopic process This challenge has drawn the attention of scientists beginning with Ramon y Cajal, who used the Golgi technique to draw detailed pictures of neurons containing most of their neurites[1] and culminates in today’s electron microscopy (EM), which allows to decode the finest details of neuronal circuit structure.[2] EM’s key advantage is the possibility to identify and distinguish presynaptic active zones containing synaptic vesicles from apposed postsynaptic structures and the visualization of even the finest axonal branches. An ideal analysis method would generate super resolution data sets, allowing to reconstruct the critical details of synaptic connectivity from intact brain samples linked to the molecular information on the types of cells and synapses and even to dynamic information on natural activity pattern history, which is causally relevant to animal behavior
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