RT Journal Article
SR Electronic
T1 <em>En bloc</em> preparation of <em>Drosophila</em> brains enables high-throughput FIB-SEM connectomics
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 855130
DO 10.1101/855130
A1 Zhiyuan Lu
A1 C. Shan Xu
A1 Kenneth J. Hayworth
A1 Patricia Rivlin
A1 Stephen M. Plaza
A1 Louis Scheffer
A1 Gerald M. Rubin
A1 Harald F. Hess
A1 Ian A. Meinertzhagen
YR 2019
UL http://biorxiv.org/content/early/2019/11/27/855130.abstract
AB Deriving the detailed synaptic connections of the entire nervous system has been a long term but unrealized goal of the nascent field of connectomics. For Drosophila, in particular, three sample preparation problems must be solved before the requisite imaging and analysis can even begin. The first is dissecting the brain, connectives, and ventral nerve cord (roughly comparable to the brain, neck, and spinal cord of vertebrates) as a single contiguous unit. Second is fixing and staining the resulting specimen, too large for previous techniques such as flash freezing, so as to permit the necessary automated segmentation of neuron membranes. Finally the contrast must be sufficient to support synapse detection at imaging speeds that enable the entire connectome to be collected. To address these issues, we report three major novel methods to dissect, fix, dehydrate and stain this tiny but complex nervous system in its entirety; together they enable us to uncover a Focused Ion-Beam Scanning Electron Microscopy (FIB-SEM) connectome of the entire Drosophila brain. They reliably recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection, for which only minimal human intervention is required. Our advanced procedures use: a custom-made jig to microdissect both regions of the central nervous system, dorsal and ventral, with their connectives; fixation and Durcupan embedment, followed by a special hot-knife slicing protocol to reduce the brain to dimensions suited to FIB; contrast enhancement by heavy metals; together with a progressive lowering of temperature protocol for dehydration. Collectively these optimize the brain’s morphological preservation, imaging it at a usual resolution of 8nm per voxel while simultaneously speeding the formerly slow rate of FIB-SEM. With these methods we could recently obtain a FIB-SEM image stack of the Drosophila brain eight times faster than hitherto, at approximately the same rate as, but without the requirement to cut, nor imperfections in, EM serial sections.