Zeqi Yu

Elucidating structure–activity relationships in semiconductor photocatalysis has been significantly impeded by the inherent limitations of ensemble-averaged characterization techniques, which obscure the spatiotemporal heterogeneity intrinsic to catalytic surfaces. Single-molecule fluorescence microscopy (SMFM) surmounts this bottleneck by offering nanometer-scale spatial resolution coupled with the capacity to resolve single-turnover events. Herein, we provide a comprehensive overview of the State-of-the-Art applications of fluorogenic probe-coupled SMFM in deciphering the microscopic mechanisms governing photocatalysis. We begin by delineating the operational principles of total internal reflection fluorescence (TIRF) microscopy and categorizing the response mechanisms of three distinct classes of fluorogenic probes: oxidative (e.g., Amplex Red, APF), reductive (e.g., Resazurin, DN-BODIPY), and acidic (e.g., furfuryl alcohol, thiophene) reporters. Subsequently, we highlight seminal studies wherein SMFM has been leveraged to visualize facet-dependent charge separation on model photocatalysts—including TiO₂, BiOBr, and InSe—to map the dynamic activity associated with surface defects and to precisely locate active sites during photoelectrochemical water splitting. Finally, we critically assess the prevailing technical challenges, such as limitations in probe specificity and background interference, while offering a perspective on prospective avenues for methodological refinement. This review is intended to serve as a methodological cornerstone for advancing mechanistic understanding in photocatalysis and for guiding the rational design of high-performance catalysts.