A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms

By X. Shu, V. Lev-Ram, Thomas J. Deerinck, Y. Qi, Ericka B. Ramko, Michael W. Davidson, Yishi Jin, Mark Ellisman, Roger Tsien. PLoS Biology, 2011

807 citations recorded by the source corpus.

Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce “miniSOG” (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.

View Paper: https://www.semanticscholar.org/paper/6741aaf1a142890ccbfee1f34a39c9b2cf077e11

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Citations in the corpus

Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy (2012, unknown): “(2) COS-7 cell expressing endoplasmic reticulum-targeted APEX-KDEL.”
ChromEMT: Visualizing 3D chromatin structure and compaction n interphase and mitotic cells (2017, methodology): “For example, miniSOG (mini singlet oxygen generator), a green fluorescent protein that photo-oxidizes DAB, has been used as a genetic tag to visualize proteins with correlated light and EM (42, 43).”
The growing and glowing toolbox of fluorescent and photoactive proteins (2016, background): “MiniSOG is an engineered variant of a FMN-binding light, oxygen, voltage (LOV) domain from Arabidopsis thaliana that generates ROS during blue light illumination [151].”
Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics (2015, background): “Furthermore, with the advent of genetically encoded markers for electron microscopy (EM) (4, 5), the continued preeminence of SR microscopy for protein-specific structural imaging at the nanoscale is no longer assured.”
Guidelines for the use and interpretation of assays for monitoring autophagy (2012, methodology): “size of GFP, provides an additional means to genetically tag proteins for CLEM analysis under conditions that are particularly suited to subsequent TEM analysis.(100) Combinatorial assays using tandem mRFP-GFP-LC3 (see Tandem mRFP/ mCherry-GFP fluorescence microscopy) along with static TEM images sh”
Volume electron microscopy for neuronal circuit reconstruction. (2012, unknown): “The development of indicators to highlight structures of interest, such as membrane-targeted HRP [31,32] or genetically encoded ﬂuorescent reactive oxygen generators for photoconver-sion [33] is geared toward selectively increasing contrast in genetically controllable ways.”
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (2012, unknown): “The mini Singlet Oxygen Generator (miniSOG) fluorescent flavoprotein, which is less than half the size of GFP, provides an additional means to genetically tag proteins for CLEM analysis under conditions that are particularly suited to subsequent TEM analysis.(87) Combinatorial assays using tandem mo”
Structural Components of Synaptic Plasticity and Memory Consolidation. (2015, background): “Spine shape and number are not necessarily dependable predictors of synapse size, location, or composition (Fiala et al. 1998; Toni et al. 2007; Bock et al. 2011; Shu et al. 2011).”