Development of Sustainable Recombinant Oxygen Scavenging Systems for Single-Molecule Superresolution Microscopy

Abstract

Single-molecule localization microscopy (SMLM) relies on enzymatic oxygen scavenging systems to suppress photobleaching and promote fluorophore photoswitching. Conventional systems depend on commercially sourced enzymes, motivating development of sustainable recombinant alternatives. This work investigated two recombinant oxygen scavenging strategies for direct stochastic optical reconstruction microscopy (dSTORM). Recombinant protocatechuate 3,4-dioxygenase (PCD)was expressed and purified but exhibited poor batch-to-batch reproducibility and limited long-term stability despite extensive biochemical optimization. These findings established reproducibility and storage stability as essential engineering requirements. Building on these observations, a fully recombinant pyranose oxidase/ascorbate peroxidase (PyOx/APEX2) system demonstrated imaging performance comparable to conventional glucose oxidase/catalase and commercial PyOx/APEX2 buffers while using substantially lower PyOx concentrations. The recombinant system exhibited robust reproducibility and supported quantitative dSTORM imaging of nuclear pore complexes and HIV-1 conical cores. Collectively, this work establishes recombinant oxygen scavenging as a practical, sustainable, and engineerable approach for superresolution microscopy.