Recent advances in single-molecule spectroscopic imaging techniques, such as spectrally resolved stochastic optical reconstruction microscopy (SR-STORM), have been effective for obtaining detailed spectral information at the molecular level. However, its application for single-molecule sensing is highly challenging owing to its complicated configuration and limited spectral information. In this study, we demonstrated single-molecule polarity sensing by combining grating-based SR-STORM with the solvatochromic dye, Nile red. The spatial and spectral resolutions of the custom-built grating-based SR-STORM were examined for various color ranges of fluorescent dyes, and this approach was successfully applied for sensing nanoscale local polarity of various organic solvent molecules and surfactant molecules. Furthermore, we demonstrated that the proposed method effectively distinguished the different polarities of functional groups within surfactant molecules at the single-molecule level. We anticipate that the proposed approach can be combined with other chromic molecules for application to many other chemical systems.

Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.

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