Hybrid labeling system for dSTORM imaging of endoplasmic reticulum for uncovering ultrastructural transformations under stress conditions.

The endoplasmic reticulum (ER) transforms its morphology to fit versatile cellular functions especially under stress conditions. Since various ER stresses are critical pathophysiological factors, the precise observations of ER can provide insights into disease diagnoses and biological researches. Live-cell super-resolution imaging is highly expected for uncovering microstructures of ER. However, to achieve this, there remains a big challenge in how to efficiently label ER with advanced fluorophores. Herein, we report a new SNAP-tag fluorescent probe, namely, CLP-TMR, for specific and high-density labeling of the newly constructed dual ER-signal (targeting and retention) peptides fused-SNAP proteins. This hybrid labeling system integrating chemical probes with genetically encoded techniques enables molecular position reconstructions of ER morphologies through direct stochastic optical reconstruction microscopy (dSTORM) imaging. The super-resolution imaging reveals several never-known ultrastructural changes responding to different ER stresses, i.e. the formation of peripheral ER sheets to restore the immunogenicity, and the long flattened ER tubules under inflammation.