Simultaneous spectroscopic pH detection and single-molecule, super-resolution fluorescence microscopy of corrosion in situ

The ability to acquire local pH information is key to understanding corrosion processes, as detection of areas with low pH can inform where active corrosion reactions are taking place. Typical methods to sense pH introduce larger, bulk scale pH sensors or use electrochemical techniques which add external factors that can influence the corrosion process. Here we probe the pH of active corrosion sights in situ by adding spectral analysis to fluorescence microscopy of nM concentrations of fluorophores that spectrally respond to corrosion reactions. Single-molecule, super-resolution microscopy is supplemented with the use of a diffraction grating to allow simultaneous measurement of single-molecule activity and the individual spectra of each molecule. While probing corrosion reduction reactions via the turn-on fluorophores resazurin and resorufin, their spectra can shift due to localized changes in pH, which can be observed in real time. We measure localized pH drops near active corrosion pits identified via super-resolution microscopy to 500 nm spatial and 20 nm spectral resolutions. These results show that it is possible to obtain localized pH information alongside simultaneous single-molecule, super-resolution microscopy of active corrosion pits in situ. In addition, correlation between pit localization via pH sensing and super-resolution microscopy techniques using the same fluorophores lends support to both techniques' ability to detect active single corrosion pits.