The combination of fluorescence light microscopy (FLM) and electron microscopy (EM) makes it possible to put molecular identity in its full ultrastructural context. With array tomography (AT) long ribbons of serial ultrathin sections are labelled via immunohistochemistry (IHC). After FLM imaging the same sections are processed for scanning electron microscopy (SEM) and re-analyzed. The resulting images are correlated and superimposed. Due to the serial nature of this approach it is possible to obtain large volumes of correlated multi-channel light and electron microscopic data. One drawback of this method is the large discrepancy of resolution between light and electron microscopy. To alleviate this we advanced AT for two super-resolution light microscopy techniques, Structured Illumination Microscopy (SIM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). We also devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. Together, these advances make it possible to map even small subcellular structures with high precision and confidence. We applied our super-resolution AT approach in C. elegans to address an important problem in connectomics research: the mapping of gap junctions at connectomes.

Figures:

Serial sections through the retrovesicular ganglion of a young adult hermaphrodite reveal UNC-7::GFP containing gap junctions. Displayed are inverted SEM images of 5 serial 100 nm sections showing neuropil tissue, corresponding SIM images of the same regions, and their overlays. White and black arrowheads point to one gap junction each, as it appears and disappears in consecutive sections of the series. Scale bar: 500 nm.

3D model of neurite projections of 10 UNC-7::GFP gap junction forming neurons and SAAD somatic region. The first section of the overall 29 of the SEM data set is shown. White arrowheads point to gap junctions (they appear red in the model). Light blue: nucleus of SAAD (shown for context). Yellowish colors: plasma membranes. Scale bar: 1 μm.

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EMC Abstracts - https://emc-proceedings.com/abstract/3d-mapping-of-subcellular-structures-with-super-resolution-array-tomography/