Optimizing photoswitching performance of organic dyes for SMLM through a single MEMS mirror

Whilst SMLM is able to localize molecules with nanometre precision it is only able to achieve this if the imaging parameters have been properly optimised. Key parameters we have investigated for optimisation are homogeneous excitation illumination and the optimal pH and thiol concentrations for photoswitching buffers. Typical SMLM experiments make use of conventional gaussian illumination modes meaning either a compromise in the excitation intensity is made due to overfilling of the objective lens, or an uneven illumination field of view (FOV) is observed which can cause intensity driven photoswitching differences in dye molecules located at different points in the FOV. We demonstrate the use of a single microelectromechanical system (MEMS) mirror as a cost-effective method to generate a flat-field of illumination across the FOV resulting in consistent SMLM metrics. We also show a workflow employing an intensity gradient through the MEMS in which we screen for optimal pH and thiol concentrations to obtain the best results for brightness and photoswitching performances of the carbocyanine dye Alexa Fluor 647. Finally, we have monitored the performance of the oxygen scavenger system based on glucose and glucose oxidase in open or closed environments, determining the amount of acidification present in prolonged imaging experiments.