Lifetime super‐resolution optical fluctuation imaging

Summary In this paper, we propose a promising super‐resolution imaging scheme in fluorescence lifetime domain (lifetime super‐resolution optical fluctuation imaging, ltSOFI). ltSOFI has the potential to obtain super‐resolution images by taking advantage of fluorescence lifetime blinking under wide‐field lifetime detection. The proof‐of‐concept for ltSOFI was demonstrated through numerical simulation of high‐order cumulant analysis on fluorescence lifetime blinking emitters. As a tentative experimental demonstration, we obtained super‐resolution lifetime imaging from time‐lapse FLIM recording of HeLa cells expressing a cAMP sensor using ltSOFI method. ltSOFI is expected to initiate a new dimension in the lifetime domain for blinking‐based super‐resolution microscopy. Lay Description We report on a promising super‐resolution imaging scheme in fluorescence lifetime domain (lifetime super‐resolution optical fluctuation imaging, ltSOFI). ltSOFI has the potential to obtain super‐resolution images by taking advantage of fluorescence lifetime blinking under wide‐field lifetime detection. Past advances in super‐resolution fluorescence microscopy primarily rely on the spatiotemporal modulation of the fluorescence intensity. Although the applications of the Q‐dot blinking have been discussed in the literature, most of the discussions have focused on the blinking of fluorescence intensity. Few studies have shown the possibility of super‐resolution imaging through fluorescence lifetime fluctuations. In this paper, we proposed the ltSOFI scheme that explored the possibility of super‐resolution reconstruction from the blinking of fluorescence lifetime. The proof‐of‐concept for ltSOFI was demonstrated through numerical simulation of high‐order cumulant analysis on fluorescence lifetime blinking emitters. As a tentative experimental demonstration, we obtained super‐resolution lifetime imaging from time‐lapse FLIM recording of HeLa cells expressing a cAMP sensor using ltSOFI method. The ltSOFI method is expected to initiate a new dimension in the lifetime domain for blinking‐based super‐resolution microscopy. Moreover, the existing fluorescence lifetime imaging microscopy and super‐resolution nanoscopy can benefit from the implementation of ltSOFI to significantly improve the imaging spatial resolution of fluorescence lifetime images. In addition, the proof‐of‐concept demonstration achieved by the numerical simulation and tentative experiment will provide a new perspective for obtaining fluorescence lifetime images with much finer details.