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WIREs Dev Biol
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The genetic encoded toolbox for electron microscopy and connectomics

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Developments in bioengineering and molecular biology have introduced a palette of genetically encoded probes for identification of specific cell populations in electron microscopy. These probes can be targeted to distinct cellular compartments, rendering them electron dense through a subsequent chemical reaction. These electron densities strongly increase the local contrast in samples prepared for electron microscopy, allowing three major advances in ultrastructural mapping of circuits: genetic identification of circuit components, targeted imaging of regions of interest and automated analysis of the tagged circuits. Together, the gains from these advances can decrease the time required for the analysis of targeted circuit motifs by over two orders of magnitude. These genetic encoded tags for electron microscopy promise to simplify the analysis of circuit motifs and become a central tool for structure‐function studies of synaptic connections in the brain. We review the current state‐of‐the‐art with an emphasis on connectomics, the quantitative analysis of neuronal structures and motifs. WIREs Dev Biol 2017, 6:e288. doi: 10.1002/wdev.288 This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes
Genetic encoded tags for electron microscopy. (a1) EM‐micrograph of a mouse retinal ganglion cell soma expressing HRP in the endoplasmic reticulum and rendered electron dense. (a2) Close‐up of a ganglion cell dendrite in the retina expressing erHRP (asterisk). (b1) EM‐micrograph of a mouse retinal ganglion cell soma expressing cytosolic APEX2 staining. (b2) Close‐up of two cytosolic APEX2 expressing processes in the inner plexiform layer (arrow and asterisk). The differential strength of the staining represents the differential amount of APEX2 expressed in each process via AAV infection. (c) EM‐micrograph showing electron‐dense labeled synaptic vesicles of POMC (proopiomelanocortin) positive neurons projecting from the hypothalamus. The labeling specificity was achieved by fusing HRP to the C terminus of vesicle‐associated membrane protein 2 (VAMP2). (d) sHRP staining at the contact site of two HEK293T cells: one cell is expressing the first subunit of sHRP fused to neurexin and the other cell the second sHRP subunit fused to neuroligin. sHRP staining can be seen at the cell‐cell junction (arrowheads), but also in internalized double membrane vesicles (arrows). (e) APX labeled processes of direction selective neurons of Drosophila (arrows). (f) Micrograph showing an electron‐dense product confined to the intercellular spaces surrounding dopaminergic dendrites (asterisks) in mouse retina. One of the processes is presynaptic to the soma of an amacrine cell (arrow). (g, h) EM‐micrographs showing dendrites of projection neurons in Drosophila labeled with miniSOG targeted to either the cytosol (g) (arrows) or mitochondria (h) (arrows). Scale bar: (a1, b1): 5 µm; (a2, c): 500 nm; (b2): 1 µm; (d): 300 nm; (f): 3 µm; (e, g, h): 1 µm.
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GETEM innovations. GETEM enable fast and unequivocal identification of genetic identified cell types in EM‐micrographs. (a) Overview resolution EM‐micrograph of a retinal section (30 nm/pixel) depicting an unlabeled (arrowhead) and an APEX positive somata (asterisk), as well as a small dendritic process (box). (b) Enlarge view of an APEX positive process. (c) The high contrast of these processes assists the reconstruction of the main cellular processes, as represented by the reconstruction of a retinal ganglion cell (J‐RGC) from a similar data set shown in panels (a) and (b). These reconstructions can be used to concentrate high‐resolution imaging efforts to particular regions of interest. (d) High‐resolution EM‐micrograph depicting APEX positive processes of an interneuron in the retina, the so‐called starburst amacrine cell. Unsupervised automatic segmentation algorithms can identify labeled processes with acceptable error rates compared to manual segmentation. Ground‐truth, manual segmentation (top); automatic segmentation (bottom). (e) Manual (left) and automatic (right) reconstruction of a starburst amacrine cell process. En face (top) and side view (bottom). Scale bars: (a): 10 µm; (b): 1 µm, (c): 50 µm; (d): 1 µm; (e): 10 µm.
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