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Fluorescence laser-scanning microscopy (FLSM) is a widely utilized tool in life-science research. In recent years, this technique has undergone a profound transformation, thanks to the introduction of novel single-photon avalanche diode (SPAD) array detectors. This study reveals the exciting possibilities of combining the SPAD array detector with single-molecule techniques. We propose a real-time single-molecule tracking architecture, where the SPAD array effortlessly localizes the molecule of interest, and the beam scanning architecture effectively maintains the molecule at the center of the microscope's detection volume. This approach enables comprehensive three-dimensional tracking throughout the entire cell, offering valuable insights into molecular nano-environments, interactions, and structural changes through fluorescence lifetime information. Furthermore, utilizing the same FLSM system, we present a novel sequential structure illumination single-molecule localization microscope (similar to MINFLUX). This advanced technique achieves localization precision in the few-nanometer range while simultaneously providing the molecule's fluorescence lifetime.
Bucci et al. (Tue,) studied this question.