Kinetic and Spatial Resolution by dSTORM of miniSOG-Mediated Proximity Labeling in Mammalian Cells.

The mini Singlet Oxygen Generator (miniSOG) is a genetically encoded flavin-binding protein that enables photocatalytic proximity labeling upon blue-light illumination. Despite its widespread adoption for proteomic and transcriptomic applications, key operational parameters such as labeling kinetics and effective spatial reach have remained undefined in cellular environments. Here, we implement a microscope-based irradiation platform coupled with single-molecule localization microscopy to quantitatively characterize miniSOG activity in fixed mammalian cells. We identify two illumination-dependent patterns: a productive window at intermediate power yielding sustained signal accumulation, and photoinactivation at higher power. Super-resolution imaging reveals that the effective labeling diameter expands with illumination time, from ∼70 nm at early illumination to ∼180 nm after 30 min, consistent across cytoskeletal and nuclear contexts and sensitive to singlet oxygen quenching. These results provide the first in situ quantification of miniSOG's kinetic and spatial capture parameters, and demonstrate that the effective proximity of labeling can be tuned to meet the needs of varying biological applications. Our findings establish a framework for rigorous experimental design and ensure that miniSOG-based proteomic and transcriptomic studies are interpreted within spatially meaningful constraints.