Automated Cyclic Super-Resolution Microscopy for Nanoscale Protein Mapping

Nanoscale mapping of multiple molecular targets is essential for decoding cellular architecture, but current approaches are limited by low throughput, intensive manual intervention, and signal variability across imaging cycles. Here, we introduce CycSTORM, an integrated super-resolution imaging platform for automated cyclic (d)STORM that enables multiplexed nanoscale protein mapping in single cells. To overcome the instability inherent in multi-day imaging, CycSTORM combines automated fluidic exchange, active 3D drift correction, and an oxygen-excluded imaging environment to stabilize fluorophore blinking and enables sub-5 nm registration across day-long experiments. Furthermore, CycSTORM incorporates a rapid chemical inactivation step using meta-chloroperoxybenzoic acid (mCPBA), eliminating >99.9% residual fluorescence within 10 minutes and minimizing inter-cycle crosstalk while preserving sample integrity. By standardizing labeling to Alexa Fluor 647, a fluorophore with stable and well-characterized blinking behavior, CycSTORM minimizes fluorophore-dependent variation and provides a robust platform for consistent localization precision across cycles, enabling reliable mapping of protein organization in single cells. Using CycSTORM, we simultaneously map multiple protein targets within the same cells with nanometer precision. Together, these advances transform cyclic super-resolution imaging into a scalable approach for quantitative nanoscale mapping of molecular organization in single cells.