In recent years, Image Scanning Microscopy (ISM) has emerged as a powerful technique for achieving super-resolution bio-imaging across various applications. Particularly noteworthy is the implementation of a single-photon detector array, enabling the utilization of Lifetime Image Scanning Microscopy, which has proven to be highly effective. In our study, we present a novel approach that combines ISM with direct Stochastic Optical Reconstruction Microscopy (dSTORM), resulting in a doubling of the localization precision in Single Molecule Localization Microscopy (SMLM). Additionally, we capitalize on the available lifetime information provided by ISM, allowing for multilabel fluorescence measurements without the detrimental effects of chromatic aberration, even at resolutions significantly surpassing the diffraction limit.
Moreover, we introduce a freely available add-on to previously employed open-source tools for single particle tracking and localization, enhancing the accessibility and utility of our methodology. This add-on serves as a valuable resource for the research community, facilitating the adoption and further advancement of the combined ISM and dSTORM technique.
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